Radiolabelled quinoline and quinolinone derivatives and their use as metabotropic glutamate receptor ligands

ABSTRACT

The present invention is concerned with radiolabelled quinoline and quinolinone derivatives according Formula (I-A)* or (I-B)* showing metabotropic glutamate receptor antagonistic activity, in particular mGlu1 receptor activity, and their preparation; it further relates to compositions comprising them, as well as their use for marking and identifying metabotropic glutamate receptor sites and for imaging an organ.  
                 
 
     In a preferable embodiment, X represents O; R 1  represents C 1-6 alkyl; cycloC 3-12 alkyl or (cycloC 3-12 alkyl)C 1-6 alkyl, wherein one or more hydrogen atoms in a C 1-6 alkyl-moiety or in a cycloC 3-12 alkyl-moiety optionally may be replaced by C 1-6 alkyloxy, aryl, halo or thienyl; R 2  represents hydrogen; halo; C 1-6 alkyl or amino; R 3  and R 4  each independently represent hydrogen or C 1-6 alkyl; or R 2  and R 3  may be taken together to form —R 2 —R 3 —, which represents a bivalent radical of formula -Z 4 -CH 2 —CH 2 —CH 2 — or -Z 4 -CH 2 —CH 2 — with Z 4  being O or NR 11  wherein R 11  is C 1-6 alkyl; and wherein each bivalent radical is optionally substituted with C 1-6 alkyl; or R 3  and R 4  may be taken together to form a bivalent radical of formula —CH 2 —CH 2 —CH 2 —CH 2 —; R 5  represents hydrogen; Y represents O; and aryl represents phenyl optionally substituted with halo. Most preferred are radiolabelled compounds in which the radioactive isotope is selected from the group of of  3 H,  11 C and  18 F. The invention also relates to their use in a diagnostic method, in perticular for marking and identifying a mGluR1 receptor in biological material, as well as to their use for imaging an organ, in particular using PET.

The present invention is concerned with radiolabelled quinoline andquinolinone derivatives showing metabotropic glutamate receptorantagonistic activity, in particular mGlu1 receptor activity, and theirpreparation; it further relates to compositions comprising them, as wellas their use in a diagnostic method, in particular for marking andidentifying metabotropic glutamate receptor sites and for imaging anorgan.

INTRODUCTION

The neurotransmitter glutamate is considered to be the major excitatoryneurotransmitter in the mammalian central nervous system. The binding ofthis neurotransmitter to metabotropic glutamate receptors (mGluRs),which are a subfamily of the G-protein-coupled receptors and whichcomprise 8 distinct subtypes of mGluRs, namely mGluR1 through mGluR8,activates a variety of intracellular second messenger systems. ThemGluRs can be divided into 3 groups based on amino acid sequencehomology, the second messenger system utilized by the receptors and thepharmacological characteristics. Group I mGluRs, which comprises mGluRsubtype 1 and 5, couple to phospholipase C and their activation leads tointracellular calcium-ion mobilization. Group II mGluRs (mGluR2 and 3)and group III mGluRs (mGluR4, 6, 7 and 8) couple to adenyl cyclase andtheir activation causes a reduction in second messenger cAMP and as sucha dampening of the neuronal activity. Treatment with Group I mGluRantagonists has been shown to translate in the parasynapsis into areduced release of neurotransmitter glutamate and to decrease theglutamate-mediated neuronal excitation via postsynaptic mechanisms.Since a variety of pathophysiologic processes and disease statesaffecting the central nervous system are thought to be due to excessiveglutamate induced excitation of the central nervous system neurons,Group I mGluR antagonists, in particular mGluR1 antagonists could betherapeutically beneficial in the treatment of central nervous systemdiseases, in particular in psychiatric and neurological diseases.

However, up to now, no specific mGluR1-ligands were available, a lackseverely hampering the study of the mGlu1 receptors, in particular theradioautographic investigations of the unequivocal distribution andabundance of these receptors in brain sections. For group 1, only[³H]glutamate was available so far, being used on rat (Thomsen et al.,Brain Res. 619:22-28, 1993) or human (Kingston et al., Neuropharmacology37:277-287, 1998) mGlu1a receptors. For the mGlu1a receptor and themGlu5 receptor [³H]quisqualate is available, however, said receptor isnot specific for the mGlu1 receptor (it also binds to the AMPA receptor)and it is competitive, i.e. it displaces glutamate (Mutel at al., J.Neurochem. 75:2590-2601, 2000).

It has been the goal of this invention to provide suitable specific, inparticular non-competitive mGlu1 receptor ligands.

The inventors have now found a particular group of compounds that—in aradiolabelled form—provides for suitable specific, in particularnon-competitive mGlu1 receptor ligands as well as a method for markingand identifying metabotropic glutamate receptor sites and for imaging anorgan.

In the framework of this application, the term “specific” means that theligand binds preferentially to the mGlu1 receptor site. The term“non-competitive” means that the ligand does not or only marginallydisplaces glutamate bonded to the mGlu1 receptor site.

WO 02/28837 discloses the non-radioactive compounds according to thepresent invention.

WO 99/26927 discloses antagonists of Group I mGluRs for treatingneurological diseases and disorders, based—among others—on a quinolinestructure.

WO 99/03822 discloses bicyclic metabotropic glutamate receptor ligands,none of them based on a quinoline or quinolinone structure.

WO 94/27605 discloses 1,2,3,4-tetrahydroquinoline-2,3,4-trione-3 or4-oximes and use thereof for treating and preventing neuronal loss,neurodegenerative diseases, adverse consequences of the hyperactivity ofthe excitatory amino acids and anxiety, as well as radiolabelledcompounds thereof.

DETAILED DESCRIPTION OF THE INVENTION

The present invention concerns the radiolabelled compounds of Formula(I-A)* or (I-B)

an N-oxide form, a pharmaceutically acceptable addition salt, aquaternary amine and a stereochemical isomeric form thereof, wherein

-   X represents O; C(R⁶)₂ with R⁶ being hydrogen, aryl or C₁₋₆alkyl    optionally substituted with amino or mono- or di(C₁₋₆alkyl)amino; S    or N—R⁷ with R⁷ being amino or hydroxy;-   R¹ represents C₁₋₆alkyl; aryl; thienyl; quinolinyl; cycloC₃₋₂alkyl    or (cycloC₃₋₁₂alkyl)C₁₋₆alkyl, wherein the cycloC₃₋₁₂alkyl moiety    optionally may contain a double bond and wherein one carbon atom in    the cycloC₃₋₁₂alkyl moiety may be replaced by an oxygen atom or an    NR⁸-moiety with R⁸ being hydrogen, benzyl or C₁₋₆alkyloxycarbonyl;    wherein one or more hydrogen atoms in a C₁₋₆alkyl-moiety or in a    cycloC₃₋₁₂alkyl-moiety optionally may be replaced by C₁₋₆alkyl,    hydroxyC₁₋₆alkyl, haloC₁₋₆alkyl, aminoC₁₋₆alkyl, hydroxy,    C₁₋₆alkyloxy, arylC₁₋₆alkyloxy, halo, C₁₋₆alkyloxycarbonyl, aryl,    amino, mono- or di(C₁₋₆alkyl)amino, C₁₋₆alkyloxycarbonylamino, halo,    piperazinyl, pyridinyl, morpholinyl, thienyl or a bivalent radical    of formula —O—, —O—CH₂—O or —O—CH₂—CH₂—O—; or a radical of formula    (a-1)    -   wherein        -   Z₁ is a single covalent bond, O, NH or CH₂;        -   Z₂ is a single covalent bond, O, NH or CH₂;        -   n is an integer of 0, 1, 2 or 3;        -   and wherein each hydrogen atom in the phenyl ring            independently may optionally be replaced by halo, hydroxy,            C₁₋₆alkyl, C₁₋₆alkyloxy or hydroxyC₁₋₆alkyl;-   or X and R¹ may be taken together with the carbon atom to which X    and R¹ are attached to form a radical of formula (b-1), (b-2) or    (b-3);-   R² represents hydrogen; halo; cyano; C₁₋₆alkyl; C₁₋₆alkyloxy;    C₁₋₆alkylthio; C₁₋₆alkylcarbonyl; C₁₋₆alkyloxycarbonyl;    C₁₋₆alkylcarbonyloxyC₁₋₆alkyl; C₂₋₆alkenyl; hydroxyC₂₋₆alkenyl;    C₂₋₆alkynyl; hydroxyC₂₋₆alkynyl; tri(C₁₋₆alkyl)silaneC₂₋₆alkynyl;    amino; mono- or di(C₁₋₆alkyl)amino; mono- or    di(C₁₋₆alkyloxyC₁₋₆alkyl)amino; mono- or    di(C₁₋₆alkylthioC₁₋₆alkyl)amino; aryl; arylC₁₋₆alkyl;    arylC₂₋₆alkynyl; C₁₋₆alkyloxyC₁₋₆alkylaminoC₁₋₆alkyl; aminocarbonyl    optionally substituted with C₁₋₆alkyl, C₁₋₆alkyloxyC₁₋₆alkyl,    C₁₋₆alkyloxycarbonylC₁₋₆alkyl or pyridinylC₁₋₆alkyl; a heterocycle    selected from thienyl, furanyl, pyrrolyl, thiazolyl, oxazolyl,    imidazolyl, isothiazolyl, isoxazolyl, pyrazolyl, pyridyl, pyrazinyl,    pyridazinyl, pyrimidinyl, piperidinyl and piperazinyl, optionally    N-substituted with C₁₋₆alkyloxyC₁₋₆alkyl, morpholinyl,    thiomorpholinyl, dioxanyl or dithianyl a radical —NH—C(═O)R⁹ wherein    R⁹ represents    -   C₁₋₆alkyl optionally substituted with cycloC₃₋₁₂alkyl,        C₁₋₆alkyloxy, C₁₋₆alkyloxycarbonyl, aryl, aryloxy, thienyl,        pyridinyl, mono- or di(C₁₋₆alkyl)amino, C₁₋₆alkylthio,        benzylthio, pyridinylthio or pyrimidinylthio;    -   cycloC₃₋₁₂alkyl; cyclohexenyl; amino; arylcycloC₃₋₁₂alkylamino;        mono- or -di(C₁₋₆alkyl)amino; mono- or        di(C₁₋₆alkyloxycarbonylC₁₋₆alkyl)amino; mono- or        di(C₁₋₆alkyloxycarbonyl)amino; mono- or di(C₂₋₆alkenyl)amino;        mono- or di(arylC₁₋₆alkyl)amino; mono- or diarylamino;        arylC₂₋₆alkenyl; furanylC₂₋₆alkenyl; piperididinyl; piperazinyl;        indolyl; furyl; benzofuryl; tetrahydrofuryl; indenyl; adamantyl;        pyridinyl; pyrazinyl; aryl; arylC₁₋₆alkylthio or a radical of        formula (a-1);    -   a sulfonamid —NH—SO₂—R¹⁰ wherein R¹⁰ represents C₁₋₆alkyl, mono-        or poly haloC₁₋₆alkyl, arylC₁₋₆alkyl, arylC₂₋₆alkenyl, aryl,        quinolinyl, isoxazolyl or di(C₁₋₆alkyl)amino;-   R³ and R⁴ each independently represent hydrogen; halo; hydroxy;    cyano; C₁₋₆alkyl; C₁₋₆alkyloxy; C₁₋₆alkyloxyC₁₋₆alkyl;    C₁₋₆alkylcarbonyl; C₁₋₆alkyloxycarbonyl; C₂₋₆alkenyl;    hydroxyC₂₋₆alkenyl; C₂₋₆alkynyl; hydroxyC₂₋₆alkynyl;    tri(C₁₋₆alkyl)silaneC₂₋₆alkynyl; amino; mono- or di(C₁₋₆alkyl)amino;    mono- or di(C₁₋₆alkyloxyC₁₋₆alkyl)amino; mono- or    di(C₁₋₆alkylthioC₁₋₆alkyl)amino; aryl; morpholinylC₁₋₆alkyl or    piperidinylC₁₋₆alkyl; or-   R² and R³ may be taken together to form —R²—R³—, which represents a    bivalent radical of formula —(CH₂)₃—, —(CH₂)₄—, —(CH₂)₅—, —(CH₂)₆—,    —CH═CH—CH═CH—, -Z₄-CH═CH—, —CH═CH-Z₄-, -Z₄-CH₂—CH₂—CH₂—,    —CH₂-Z₄-CH₂—CH₂—, —CH₂—CH₂-Z₄-CH₂—, —CH₂—CH₂—CH₂-Z₄-, -Z₄-CH₂—CH₂—,    —CH₂-Z₄-CH₂— or —CH₂—CH₂-Z₄-, with Z₄ being O, S, SO₂ or NR¹¹    wherein R¹¹ is hydrogen, C₁₋₆alkyl, benzyl or C₁₋₆alkyloxycarbonyl;    and wherein each bivalent radical is optionally substituted with    C₁₋₆alkyl.-   or R³ and R⁴ may be taken together to form a bivalent radical of    formula —CH═CH—CH═CH— or —CH₂—CH₂—CH₂—CH₂—;-   R⁵ represents hydrogen; cycloC₃₋₁₂alkyl; piperidinyl; oxo-thienyl;    tetrahydrothienyl, arylC₁₋₆alkyl; C₁₋₆alkyloxyC₁₋₆alkyl;    C₁₋₆alkyloxycarbonylC₁₋₆alkyl or C₁₋₆alkyl optionally substituted    with a radical C(═O)NR_(x)R_(y), in which R_(x) and R_(y), each    independently are hydrogen, cycloC₃₋₁₂alkyl, C₂₋₆alkynyl or    C₁₋₆alkyl optionally substituted with cyano, C₁₋₆alkyloxy,    C₁₋₆alkyloxycarbonyl, furanyl, pyrrolidinyl, benzylthio, pyridinyl,    pyrrolyl or thienyl;-   Y represents O or S;-   or Y and R⁵ may be taken together to form ═Y—R⁵— which represents a    radical of formula    —CH═N—N═  (c-1);    —N═N—N═  (c-2); or    —N—CH═CH—  (c-3);-   aryl represents phenyl or naphthyl optionally substituted with one    or more substituents selected from halo, hydroxy, C₁₋₆alkyl,    C₁₋₆alkyloxy, phenyloxy, nitro, amino, thio, C₁₋₆alkylthio,    haloC₁₋₆alkyl, polyhaloC₁₋₆alkyl, polyhaloC₁₋₆alkyloxy,    hydroxyC₁₋₆alkyl, C₁₋₆alkyloxyC₁₋₆alkyl, aminoC₁₋₆alkyl, mono- or    di(C₁₋₆alkyl)amino; mono- or di(C₁₋₆alkyl)aminoC₁₋₆alkyl, cyano,    —CO—R¹², —CO—OR¹³, —NR¹³SO₂R¹², —SO₂—NR¹³R¹⁴, —NR¹³C(O)R¹²,    —C(O)NR¹³R¹⁴, —SOR¹², —SO₂R¹²; wherein each R¹², R¹³ and R¹⁴    independently represent C₁₋₆alkyl; cycloC₃₋₆alkyl; phenyl; phenyl    substituted with halo, hydroxy, C₁₋₆alkyl, C₁₋₆alkyloxy,    haloC₁₋₆alkyl, polyhaloC₁₋₆alkyl, furanyl, thienyl, pyrrolyl,    imidazolyl, thiazolyl or oxazolyl;    and when the R¹—C(═X) moiety is linked to another position than the    7 or 8 position, then said 7 and 8 position may be substituted with    R¹⁵ and R¹⁶ wherein either one or both of R¹⁵ and R¹⁶ represents    C₁₋₆alkyl, C₁₋₆alkyloxy or R¹⁵ and R¹⁶ taken together may form a    bivalent radical of formula —CH═CH—CH═CH—.

As used in the foregoing definitions and hereinafter C₁₋₆alkyl as agroup or part of a group encompasses the straight and branched chainsaturated hydrocarbon radicals having from 1 to 6 carbon atoms such as,for example, methyl, ethyl, propyl, butyl, pentyl or hexyl; C₂₋₆alkenylas a group or part of a group encompasses the straight and branchedchain hydrocarbon radicals having from 2 to 6 carbon atoms and having adouble bond such as ethenyl, propenyl, butenyl, pentenyl, hexenyl,3-methylbutenyl and the like; C₂₋₆alkynyl as a group or part of a groupdefines straight or branched chain hydrocarbon radicals having from 2 to6 carbon atoms and having a triple bond such as ethynyl, propynyl,butynyl, pentynyl, hexynyl, 3-methylbutynyl and the like; cycloC₃₋₆alkylencompasses monocyclic alkyl ring structures such as cyclopropyl,cyclobutyl, cyclopentyl, and cyclohexyl; cycloC₃₋₁₂alkyl encompassesmono-, bi- or tricyclic alkyl ring structures and is generic to forexample cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl,cyclooctyl, norbornanyl, adamantyl.

The term halo is generic to fluoro, chloro, bromo and iodo. As used inthe foregoing and hereinafter, polyhaloC₁₋₆alkyl as a group or part of agroup is defined as mono- or polyhalosubstituted C₁₋₆alkyl, inparticular methyl with one or more fluoro atoms, for example,difluoromethyl or trifluoromethyl. In case more than one halogen atomsare attached to an alkyl group within the definition ofpolyhaloC₁₋₆alkyl, they may be the same or different.

When any variable, e.g. aryl, occurs more than one time in anyconstituent, each definition is independent.

When any bond is drawn into a ring structure, it means that thecorresponding substituent may be linked to any atom of said ringstructure. This means for instance that the R¹—C(═X) moiety may belinked to the quinoline or quinolinone moiety in position 5, 6, 7, 8 butalso position 3 or position 4.

By the term “radiolabelled compound” is meant any compound according toFormula (I-A)* or (I-B)*, an N-oxide form, a pharmaceutically acceptableaddition salt, a quaternary amine or a stereochemically isomeric formthereof, which contains at least one radioactive atom. In the frameworkof this application, compounds which do not contain a radio-active atomare denoted without an asterisk to their formula number, compounds whichcontain a radio-active atom are denoted with an asterisk to theirformula number. Compounds can be labelled with either positron or gammaemitting radionuclides. For radioligand-binding techniques (membranereceptor assay), the [³H]-atom or the [¹²⁵I]-atom is the atom of choice.For imaging, the most commonly used positron emitting (PET)radionuclides are ¹¹C, ¹⁸F, ¹⁵O and ¹³N, all of which are acceleratorproduced and have half-lives of 20, 100, 2 and 10 minutes respectively.Since the half-lives of these radionuclides are so short, it is onlyfeasible to use them at institutions which have an accelerator on sitefor their production, thus limiting their use. The most widely used ofthese are ¹⁸F, ^(99m)Tc, ²⁰¹Tl and ¹²³I.

In particular, the radioactive atom is selected from the group ofhydrogen, carbon, nitrogen, sulfur, oxygen and halogen. Preferably, theradioactive atom is selected from the group of hydrogen, carbon andhalogen.

In particular, the radioactive atom is selected from the group of ³H,¹¹C, ¹⁸F, ¹²²I, ¹²³I, ¹²⁵I, ¹³¹I, ⁷⁵Br, ⁷⁶Br, ⁷⁷Br and ⁸²Br. Preferably,the radioactive atom is selected from the group of ³H, ¹¹C and ¹⁸F.

By the term “compound according to the invention” is meant a compoundaccording to Formula (I-A)* or (I-B)*, an N-oxide form, apharmaceutically acceptable addition salt, a quaternary amine and astereochemically isomeric form thereof.

For in vivo use, salts of the compounds of Formula (I-A)* and (I-B)* arethose wherein the counter ion is pharmaceutically acceptable. However,salts of acids and bases which are non-pharmaceutically acceptable mayalso find use, for example, in the preparation or purification of apharmaceutically acceptable compound. All salts, whetherpharmaceutically acceptable or not are included within the ambit of thepresent invention. With the term “in vivo” is meant any use of thecompounds according to the invention whereby said compounds areadministered to live animals.

The pharmaceutically acceptable addition salts as mentioned hereinaboveare meant to comprise the therapeutically active non-toxic acid additionsalt forms which the compounds of Formula (I-A)* and (I-B)* are able toform. The latter can conveniently be obtained by treating the base formwith such appropriate acids as inorganic acids, for example, hydrohalicacids, e.g. hydrochloric, hydrobromic and the like; sulfuric acid;nitric acid; phosphoric acid and the like; or organic acids, forexample, acetic, propanoic, hydroxyacetic, 2-hydroxypropanoic,2-oxopropanoic, oxalic, malonic, succinic, maleic, fumaric, malic,tartaric, 2-hydroxy-1,2,3-propanetricarboxylic, methanesulfonic,ethanesulfonic, benzenesulfonic, 4-methylbenzenesulfonic,cyclohexanesulfamic, 2-hydroxybenzoic, 4-amino-2-hydroxybenzoic and thelike acids. Conversely the salt form can be converted by treatment withalkali into the free base form.

The compounds of Formula (I-A)* and (I-B)* containing acidic protons maybe converted into their therapeutically active non-toxic metal or amineaddition salt forms by treatment with appropriate organic and inorganicbases. Appropriate base salt forms comprise, for example, the ammoniumsalts, the alkali and earth alkaline metal salts, e.g. the lithium,sodium, potassium, magnesium, calcium salts and the like, salts withorganic bases, e.g. primary, secondary and tertiary aliphatic andaromatic amines such as methylamine, ethyl amine, propyl amine,isopropylamine, the four butylamine isomers, dimethylamine,diethylamine, diethanolamine, dipropylamine, diisopropylamine,di-n-butylamine, pyrrolidine, piperidine, morpholine, trimethylamine,triethylamine, tripropylamine, quinuclidine, pyridine, quinoline andisoquinoline, the benzathine, N-methyl-D-glucamine,2-amino-2-(hydroxymethyl)-1,3-propanediol, hydrabamine salts, and saltswith amino acids such as, for example, arginine, lysine and the like.Conversely the salt form can be converted by treatment with acid intothe free acid form.

The term “addition salt” also comprises the hydrates and solventaddition forms which the compounds of Formula (I-A)* and (I-B)* are ableto form. Examples of such forms are e.g. hydrates, alcoholates and thelike.

The term “quaternary amine” as used hereinbefore defines the quaternaryammonium salts which the compounds of Formula (I-A)* and (I-B)* are ableto form by reaction between a basic nitrogen of a compound of Formula(I-A)* or (I-B)* and an appropriate quaternizing agent, such as, forexample, an optionally substituted alkylhalide, arylhalide orarylalkylhalide, e.g. methyliodide or benzyliodide. Other reactants withgood leaving groups may also be used, such as alkyltrifluoromethanesulfonates, alkyl methanesulfonates, and alkylp-toluenesulfonates. A quaternary amine has a positively chargednitrogen. Pharmaceutically acceptable counter ions include chloro,bromo, iodo, trifluoroacetate and acetate. The counter ion of choice canbe introduced using ion exchange resins.

It will be appreciated that some of the compounds according to theinvention may contain one or more centers of chirality and exist asstereochemically isomeric forms.

The term “stereochemically isomeric forms” as used hereinbefore definesall the possible stereoisomeric forms which the compounds according tothe invention or physiologically functional derivatives may possess.Unless otherwise mentioned or indicated, the chemical designation ofcompounds denotes the mixture of all possible stereoisomeric forms, saidmixtures containing all diastereomers and enantiomers of the basicmolecular structure as well as each of the individual isomeric forms ofthe compounds according to the invention, substantially free, i.e.associated with less than 10%, preferably less than 5%, in particularless than 2% and most preferably less than 1% of the other isomers.Stereochemically isomeric forms of the compounds according to theinvention are obviously intended to be embraced within the scope of thepresent invention. The same applies to the intermediates as describedherein, used to prepare end products of the compounds according to theinvention.

The terms cis and trans are used herein in accordance with ChemicalAbstracts nomenclature.

In some compounds according to the invention and in the intermediatesused in their preparation, the absolute stereochemical configuration hasnot been determined. In these cases, the stereoisomeric form which wasfirst isolated is designated as “A” and the second as “B”, withoutfurther reference to the actual stereochemical configuration. However,said “A” and “B” stereoisomeric forms can be unambiguously characterizedby physicochemical characteristics such as their optical rotation incase “A” and “B” have an enantiomeric relationship. A person skilled inthe art is able to determine the absolute configuration of suchcompounds using art-known methods such as, for example, X-raydiffraction. In case “A” and “B” are stereoisomeric mixtures, they canbe further separated whereby the respective first fractions isolated aredesignated “A1” and “B1” and the second as “A2” and “B2”, withoutfurther reference to the actual stereochemical configuration.

The N-oxide forms of the present compounds are meant to comprise thecompounds of formula (I-A)* and (I-B)* wherein one or several nitrogenatoms are oxidized to the so-called N-oxide.

Some of the compounds according to the invention may also exist in theirtautomeric form. Such forms although not explicitly indicated in theabove formula are intended to be included within the scope of thepresent invention. Of special interest are those compounds of formula(I-A)* and (I-B)* which are stereochemically pure.

An interesting group of compounds are those compounds of formula (I-A)*and (I-B)* wherein

-   X represents O; C(R⁶)₂ with R⁶ being hydrogen or aryl; or N—R⁷ with    R⁷ being amino or hydroxy;-   R¹ represents C₁₋₆alkyl, aryl; thienyl; quinolinyl; cycloC₃₋₁₂alkyl    or (cycloC₃₋₁₂alkyl)C₁₋₆alkyl, wherein the cycloC₃₋₁₂alkyl moiety    optionally may contain a double bond and wherein one carbon atom in    the cycloC₃₋₁₂alkyl moiety may be replaced by an oxygen atom or an    NR⁸-moiety with R⁸ being benzyl or C₁₋₆alkyloxycarbonyl; wherein one    or more hydrogen atoms in a C₁₋₆alkyl-moiety or in a    cycloC₃₋₁₂alkyl-moiety optionally may be replaced by C₁₋₆alkyl,    haloC₁₋₆alkyl, hydroxy, C₁₋₆alkyloxy, arylC₁₋₆alkyloxy, halo, aryl,    mono- or di(C₁₋₆alkyl)amino, C₁₋₆alkyloxycarbonylamino, halo,    piperazinyl, pyridinyl, morpholinyl, thienyl or a bivalent radical    of formula —O— or —O—CH₂—CH₂—O—; or a radical of formula (a-1)    -   wherein        -   Z₁ is a single covalent bond, O or CH₂;        -   Z₂ is a single covalent bond, O or CH₂;        -   n is an integer of 0, 1, or 2;        -   and wherein each hydrogen atom in the phenyl ring            independently may optionally be replaced by halo or hydroxy;    -   or X and R¹ may be taken together with the carbon atom to which        X and R¹ are attached to form a radical of formula (b-1), (b-2)        or (b-3);-   R² represents hydrogen; halo; cyano; C₁₋₆alkyl; C₁₋₆alkyloxy;    C₁₋₆alkylthio; C₁₋₆alkylcarbonyl; C₁₋₆alkyloxycarbonyl; C₂₋₆alkenyl;    hydroxyC₂₋₆alkenyl; C₂₋₆alkynyl; hydroxyC₂₋₆alkynyl;    tri(C₁₋₆alkyl)silaneC₂₋₆alkynyl; amino; mono- or di(C₁₋₆alkyl)amino;    mono- or di(C₁₋₆alkyloxyC₁₋₆alkyl)amino; mono- or    di(C₁₋₆alkylthioC₁₋₆alkyl)amino; aryl; arylC₁₋₆alkyl;    arylC₂₋₆alkynyl; C₁₋₆alkyloxyC₁₋₆alkylaminoC₁₋₆alkyl;    -   aminocarbonyl optionally substituted with        C₁₋₆alkyloxycarbonylC₁₋₆alkyl; a heterocycle selected from        thienyl, furanyl, thiazolyl and piperidinyl, optionally        N-substituted with morpholinyl or thiomorpholinyl;    -   a radical —NH—C(═O)R⁹ wherein R⁹ represents C₁₋₆alkyl optionally        substituted with cycloC₃₋₁₂alkyl, C₁₋₆alkyloxy,        C₁₋₆alkyloxycarbonyl, aryl, aryloxy, thienyl, pyridinyl, mono-        or di(C₁₋₆alkyl)amino, C₁₋₆alkylthio, benzylthio, pyridinylthio        or pyrimidinylthio; cycloC₃₋₁₂alkyl; cyclohexenyl; amino;        arylcycloC₃₋₁₂alkylamino; mono- or -di(C₁₋₆alkyl)amino; mono- or        di(C₁₋₆alkyloxycarbonylC₁₋₆alkyl)amino; mono- or        di(C₁₋₆alkyloxycarbonyl)amino; mono- or di(C₂₋₆alkenyl)amino;        mono- or di(arylC₁₋₆alkyl)amino; mono- or diarylamino;        arylC₂₋₆alkenyl; furanylC₂₋₆alkenyl; piperididinyl; piperazinyl;        indolyl; furyl; benzofuryl; tetrahydrofuryl; indenyl; adamantyl;        pyridinyl; pyrazinyl; aryl or a radical of formula (a-1);    -   a sulfonamid —NH—SO₂—R¹⁰ wherein R¹⁰ represents C₁₋₆alkyl, mono-        or poly haloC₁₋₆alkyl, arylC₁₋₆alkyl or aryl;-   R³ and R⁴ each independently represent hydrogen; C₁₋₆alkyl;    C₁₋₆alkyloxyC₁₋₆alkyl; C₁₋₆alkyloxycarbonyl; or-   R² and R³ may be taken together to form —R²—R³—, which represents a    bivalent radical of formula —(CH₂)₄—, —(CH₂)₅—, -Z₄-CH═CH—,    -Z₄-CH₂—CH₂—CH₂— or -Z₄-CH₂—CH₂—, with Z₄ being O, S, SO₂ or NR¹¹    wherein R¹¹ is hydrogen, C₁₋₆alkyl, benzyl or C₁₋₆alkyloxycarbonyl;    and wherein each bivalent radical is optionally substituted with    C₁₋₆alkyl;-   or R³ and R⁴ may be taken together to form a bivalent radical of    formula —CH═CH—CH═CH— or —CH₂—CH₂—CH₂—CH₂—;-   R⁵ represents hydrogen; piperidinyl; oxo-thienyl; tetrahydrothienyl,    arylC₁₋₆alkyl; C₁₋₆alkyloxycarbonylC₁₋₆alkyl or C₁₋₆alkyl optionally    substituted with a radical C(═O)NR_(x)R_(y), in which R_(x) and    R_(y), each independently are hydrogen, cycloC₃₋₁₂alkyl, C₂₋₆alkynyl    or C₁₋₆alkyl optionally substituted with cyano, C₁₋₆alkyloxy or    C₁₋₆alkyloxycarbonyl;-   Y represents O or S;-   or Y and R⁵ may be taken together to form ═Y—R⁵— which represents a    radical of formula    —CH═N—N═  (c-1); or    —N═N—N═  (c-2);-   aryl represents phenyl or naphthyl optionally substituted with one    or more substituents selected from halo, C₁₋₆alkyloxy, phenyloxy,    mono- or di(C₁₋₆alkyl)amino and cyano;-   and when the R¹—C(═X) moiety is linked to another position than the    7 or 8 position, then said 7 and 8 position may be substituted with    R¹ and R¹⁶ wherein either one or both of R¹⁵ and R¹⁶ represents    C₁₋₆alkyl or R¹⁵ and R¹⁶ taken together may form a bivalent radical    of formula —CH═CH—CH═CH—.

A further most interesting group of compounds comprises those compoundsof formula (I-A)* and (I-B)* wherein X represents O;

-   R¹ represents C₁₋₆alkyl; cycloC₃₋₁₂alkyl or    (cycloC₃₋₁₂alkyl)C₁₋₆alkyl, wherein one or more hydrogen atoms in a    C₁₋₆alkyl-moiety or in a cycloC₃₋₁₂alkyl-moiety optionally may be    replaced by C₁₋₆alkyloxy, aryl, halo or thienyl;-   R² represents hydrogen; halo; C₁₋₆alkyl or amino;-   R³ and R⁴ each independently represent hydrogen or C₁₋₆alkyl; or-   R² and R² may be taken together to form —R²—R³—, which represents a    bivalent radical of formula -Z₄-CH₂—CH₂—CH₂— or -Z₄-CH₂—CH₂— with Z₄    being O or NR¹¹ wherein    -   R¹¹ is C₁₋₆alkyl; and wherein each bivalent radical is        optionally substituted with C₁₋₆alkyl;    -   or R³ and R⁴ may be taken together to form a bivalent radical of        formula —CH₂—CH₂—CH₂—CH₂—;-   R⁵ represents hydrogen;-   Y represents O; and-   aryl represents phenyl optionally substituted with halo.

A further interesting group of compounds comprises those compounds offormula (I-A)* and (I-B)* wherein the R¹—C(═X) moiety is linked to thequinoline or quinolinone moiety in position 6.

Especially interesting are the following radioactive compounds

All compounds according to the invention show a moderate to strongmGluR1 activity. Such activity is among others attributed to thespecific binding of said compound to the mGlu1 receptor, which makes thecompounds useful in a diagnostic method, e.g. for labeling and detectingmGlu1 receptor sites. The invention therefore also relates to aradiolabelled compound according to the invention for use in adiagnostic method.

FIRST PREFERRED EMBODIMENT Gamma Emitting Radionuclide

According to a first preferred embodiment, the radiolabelled compoundcomprises at least one [³H]-atom or one [¹²⁵I]-atom. A [³H]-atom isconveniently introduced by partially or completely substituting one ormore non-radioactive [³H]-hydrogen atoms in the molecule by theirradioactive isotopes. The choice of whether a [³H] or [¹²⁵I] radioligandwill be used may depend in part on the availability of liquidscintillation counters (LSC), which are fairly expensive. [¹²⁵I] ligandscan be quantified using either a γ-counter or an LSC, whereas [³H]ligands necessitate the use of an LSC.

The radiolabelled compound comprising at least one [³H]-atom or one[¹²⁵I]-atom is advantageously used in radioligand-binding techniques, inparticular in in vitro membrane receptor assays for marking oridentifying a mGlu1 receptor in biological material.

The radiolabelled compounds comprising at least one [³H]-atom or one[¹²⁵I]-atom is also advantageously used in in vivo mGlu1 receptorautoradiography of the brain since the compounds according to theinvention have the advantageous and unexpected ability to readily crossthe blood-brain barrier.

The invention therefore also relates to a radiolabelled compoundaccording to the invention used in a diagnostic method which consists ofmarking or identifying a mGlu1 receptor in biological material, as wellas the use of the compounds according to the invention for themanufacture of a diagnostic tool for marking or identifying an mGlu1receptor in biological material, whether in vivo or in vitro.

In the framework of this application, by the term “biological material”is meant to include any material which has a biological origin. Inparticular, this relates to tissue samples, plasma fluids, body fluids,body parts and organs originating from warm-blooded animals andwarm-blooded animals per se, in particular humans.

Basic experiments that are performed using the membrane assay system formarking or identifying a mGlu1 receptor in biological material are:saturation experiments, inhibition experiments, association kineticexperiments and dissociation kinetic experiments. These methods areapplicable to most neurotransmitter and hormone receptor systems,including the mGluR1-system (Methods for Neurotransmitter ReceptorAnalysis, ed. by Henry I. Yamamura et al., Raven Press Ltd., New York,1990). To this end, the radiolabelled compound is administered to thebiological material to mark the mGlu1 receptors and the emissions fromthe radiolabelled compound are detected to identify the amount orlocation of the mGlu1 receptors, for instance for ex vivo receptorautoradiography.

The radiolabelled compounds according to the invention comprising atleast one [³H]-atom or one [¹²⁵I]-atom are also useful as agents forscreening whether a test compound has the ability to occupy or bind to amGlu1 receptor site. The degree to which a test compound will displace acompound according to the invention from the mGlu1 receptor site willshow the test compound ability to occupy or bind to a mGlu1 receptor andtherefore act as either an agonist, an antagonist or a mixedagonist/antagonist of a mGlu1 receptor.

The radiolabelled compounds according to the invention comprising atleast one [³H]-atom or one [¹²⁵I]-atom are advantageously prepared bysubstituting a halogen atom with a tritium atom, as is documented in theExperimental Section below.

SECOND PREFERRED EMBODIMENT Positron Emitting Radionuclide

In a second preferred embodiment, the radiolabelled compound comprisesat least one radioactive carbon or halogen atom. In principle, anycompound according to Formula (I) containing a carbon or halogen atom isprone for radiolabel ling by replacing the carbon or halogen atom by asuitable radioactive isotope or by making the compounds according toFormula (I) using radioactively-labelled reagentia. Suitable halogenradioisotopes to this purpose are radioactive carbon, e.g. [¹¹C];radioactive iodides, e.g. [¹²²I], [¹²³I], [¹³¹I]; radioactive bromides,e.g. [⁷⁵Br], [⁷⁶Br], [⁷⁷Br] and [⁸²Br]; and radioactive fluorides, e.g.[¹⁸F]. Preferred radiolabelled compounds are those compounds of Formula(I-A)* and (I-B)*, wherein R¹ comprises a radioactive carbon or haloatom, especially [¹¹C], [¹⁸F], [¹²³I], [⁷⁵Br], [⁷⁶Br] or [⁷⁷Br].

Preparation of the Radioactive Compounds

The introduction of a radioactive halogen atom can be performed by asuitable reaction such as depicted below

in which all substituents in Formula (I-A-a) and (I-A-a)* are defined asin Formula (I-A)* and halo is a halogen atom. A suitable compound(I-A-a) is reacted with H[¹⁸F] such that the halogen atom present on thequinoline ring is displaced by a nucleophilic displacement reaction withthe radioactive [¹⁸F] atom.

For obtaining radiolabelled compounds according to Formula (I-B)*,radiolabel ling can be performed on an equivalent way, for instance byway of a reaction scheme as depicted below. Obviously, also compoundsaccording to Formula (I-A)* can be obtained in an equivalent way, i.e.by way of labelling an R¹ substituent.

Other methods for tritium-labelling are disclosed e.g. by Peng et al.Fusion Technology, American Nuclear Society, 21(2):307-311, 1992 and byBrundish et al. Journal of Labelled Compounds and Radiopharmaceuticals25(12):1361-1369 (1988).

The introduction of a radioactive [¹¹C] can be performed using thereaction scheme below in which a suitable compound (I-A-a) is firststanyllated after which the radioactive [¹¹C] is introduced using e.g. apalladium catalyzed “Stille-type” coupling reaction using[¹¹C]methyliodide (Scott, W. J.; Crisp, G. T.; Stille, J. K. J. Am.Chem. Soc., 1984, 106, 4630).

In Formula (I-A-a), (I-A-b) and (I-A-c)*, all substituents have the samemeaning as defined in Formula (I-A)*, halo is a halogen atom and R¹⁷ ismethyl or butyl.

Because of their unexpected property to penetrate readily theblood-brain barrier, the radiolabelled compounds comprising aradioactive halogen atom are advantageously administered in vivo, in anappropriate composition to an animal, especially a warm-blooded animal,and the location of said radiolabelled compounds is detected usingimaging techniques, such as, for instance, Single Photon EmissionComputered Tomography (SPECT) or Positron Emission Tomography (PET) andthe like. In this manner the distribution of mGlu1 receptor sitesthroughout the body can be detected and organs containing mGlu1 receptorsites such as, for example, the brain, can be visualized by the imagingtechniques mentioned hereinabove. This process of imaging an organ byadministering a radiolabelled compound of Formula (I-A)* or (I-A)*,which bind to the mGlu1 receptor sites and detecting the emissions fromthe radioactive compound also constitutes an aspect of the presentinvention.

The application of the compounds of Formula (I-A)* and (I-B)* in theabove described techniques constitutes a further aspect of the presentinvention. The invention in particular relates to the use of thecompounds according to the invention for the manufacture of a diagnostictool for use in PET. For use in PET, most preferred are radiolabelledcompounds according to the invention, in which a ¹⁸F is incorporated(U.S. Pat. No. 4,931,270 by Horn et al., published Jun. 5, 1990).

Preparation of the Non-Radioactive Compounds

The non-radioactive compounds according to the invention may be producedin a number of ways.

In order to simplify the structural representation of some of thepresent compounds and intermediates in the following preparationprocedures, the quinoline or the quinolinone moiety will hereinafter berepresented by the symbol Q.

The compounds of formula (I-A) or (I-B), wherein X represents O, saidcompounds being represented by formula (I_(A/B)-a), can be prepared byoxidizing an intermediate of formula (II) in the presence of a suitableoxidizing agent, such as potassium permanganate, and a suitablephase-transfer catalyst, such as tris(dioxa-3,6-heptyl)amine, in asuitable reaction-inert solvent, such as for example dichloromethane.

Compounds of formula (I_(A/B)-a) may also be prepared by reacting anintermediate of formula (III) with an intermediate of formula (IV),wherein W₁ represents a halo atom, e.g. bromo, in the presence of butyllithium and a suitable reaction-inert solvent, such as for exampletetrahydrofuran.

Alternatively, compounds of formula (I_(A/B)-a) may also be prepared byreacting an intermediate of formula (V) with an intermediate of formula(IV) in the presence of butyl lithium and a suitable reaction-inertsolvent, such as for example tetrahydrofuran.

Compounds of formula (I_(A/B)-a), wherein the R¹ substituent is linkedto the carbonyl moiety via an oxygen atom, said R¹ substituent beingrepresented by O—R^(1a) and said compounds by formula (I_(A/B)-a-1), canbe prepared by reacting an intermediate of formula (VI) with anintermediate of formula (VII) in the presence of a suitable acid, suchas sulfuric acid.

Compounds of formula (I-A), wherein R² represents methylcarbonyl, saidcompounds being represented by formula (I-A-1), can be prepared byreacting an intermediate of formula (VIII) in the presence of a suitableacid, such as hydrochloric acid, and a suitable reaction-inert solvent,such as for example tetrahydrofuran.

The compounds of formula (I) may also be converted into each otherfollowing art-known transformations.

Compounds of formula (I-A) wherein R² is a halo atom, such as chloro,can be converted into a compound of formula (I-A), wherein R² is anotherhalo atom, such as fluoro or iodo, by reaction with a suitablehalogenating agent, such as for example potassium fluoride or sodiumiodide, in the presence of a suitable reaction-inert solvent, e.g.dimethyl sulfoxide or acetonitrile and optionally in the presence ofacetyl chloride.

Compounds of formula (I-A), wherein R² is a suitable leaving group, suchas a halo atom, e.g. chloro, iodo, said leaving group being representedby W² and said compounds by (I-A-2), can be converted into a compound offormula (I-A) wherein R² is cyano, said compound being represented byformula (I-A-3), by reaction with a suitable cyano-introducing agent,such as for example trimethylsilanecarbonitrile, in the presence of asuitable base such as N,N-diethylethanamine and a suitable catalyst,such as for example tetrakis(triphenylphosphine)palladium.

Compounds of formula (I-A-2) can also be converted into a compound offormula (I-A-4) by reaction with C₂₋₆alkynyltri(C₁₋₆alkyl)silane in thepresence of CuI, an appropriate base, such as for exampleN,N-diethylethanamine, and an appropriate catalyst, such as for exampletetrakis(triphenylphosphine)palladium. Compounds of formula (I-A-4) canon their turn be converted into a compound of formula (I-A-5) byreaction with potassium fluoride in the presence of a suitable acid suchas acetic acid, or by reaction with a suitable base, such as potassiumhydroxide, in the presence of a suitable reaction-inert solvent, such asan alcohol, e.g. methanol and the like.

Compounds of formula (I-A-2) can also be converted into a compound offormula (I-A-6) by reaction with an intermediate of formula (IX) in thepresence of CuI, a suitable base, such as for exampleN,N-diethylethanamine, and a suitable catalyst such astetrakis(triphenylphosphine)palladium.

Compounds of formula (I-A-2) can also be converted into a compoundwherein R² is C₁₋₆alkyl, said compound being represented by formula(I-A-8) in the presence of a suitable alkylating agent, such as forexample Sn(C₁₋₆alkyl)₄, or into a compound wherein R² is C₂₋₆alkenyl,said compound being represented by formula (I-A-9) in the presence of asuitable alkenylating agent, such as for example Sn(C₂₋₆alkenyl)(C₁₋₆alkyl)₃, both reactions in the presence of a suitable catalyst,such as for example tetrakis(triphenylphosphine)palladium and areaction-inert solvent, such as for example toluene or dioxane.

Compounds of formula (I-A-2) can also be converted into a compound offormula (I-A-7) wherein Z represents O or S, by reaction with anintermediate of formula (X) optionally in the presence of a suitablebase such as dipotassium carbonate and a reaction-inert solvent, such asN,N-dimethyl formamide.

Compounds of formula (I-A-2) can also be converted into a compound offormula (I-A), wherein R² is C₁₋₆alkyloxycarbonyl, said compound beingrepresented by formula (I-A-10) and a compound of formula (I-A), whereinR² is hydrogen, said compound being represented by formula (I-A-11), byreaction with a suitable alcohol of formula C₁₋₆alkylOH and CO in thepresence of a suitable catalyst, such as for examplepalladium(II)acetate, triphenylphosphine, a suitable base such asdipotassium carbonate and a reaction-inert solvent, such asN,N-dimethylformamide.

Compounds of formula (I-A-11) can also be prepared by reacting acompound of formula (I-A-2) with Zn in the presence of a suitable acidsuch as acetic acid.

Compounds of formula (I-A-2) can also be converted into a compound offormula (I-A), wherein R² is aminocarbonyl substituted withC₁₋₆alkyloxycarbonylC₁₋₆alkyl, said compound being represented byformula (I-A-12), by reaction with an intermediate of formulaH₂N—C₁₋₆alkyl-C(═O)—O—C₁₋₆alkyl in the presence of CO, a suitablecatalyst such as tetrakis(triphenylphosphine)palladium, a suitable base,such as for example N,N-diethylethanamine, and a suitable reaction-inertsolvent, such as for example toluene.

Compounds of formula (I-A-2) can also be converted into a compound offormula (I-A) wherein R² is aryl or a heterocycle selected from thegroup described in the definition of R² hereinabove, said R² beingrepresented by R^(2a) and said compound by formula (I-A-13) by reactionwith an intermediate of formula (XI), (XII) or (XIII) in the presence ofa suitable catalyst such as for exampletetrakis(triphenylphosphine)palladium and a suitable reaction-inertsolvent, such as for example dioxane.

Compounds of formula (I-A-2) can also be converted into a compound offormula (I-B), wherein Y and R⁵ are taken together to form a radical offormula (b-1) or (b-2), said compound being represented by formula(I-B-1) or (I-B-2), by reaction with hydrazincarboxaldehyde or sodiumazide in a suitable reaction-inert solvent, such as an alcohol, e.g.butanol, or N,N-dimethylformamide.

Compounds of formula (I-A-11) can be converted into the correspondingN-oxide, represented by formula (I-A-14), by reaction with a suitableperoxide, such as 3-chloro-benzenecarboperoxoic acid, in a suitablereaction-inert solvent, such as for example methylene chloride. Saidcompound of formula (I-A-14) can further be converted into a compound offormula (I-B), wherein R⁵ is hydrogen, said compound being representedby formula (I-B-3), by reaction with 4-methyl-benzene sulfonyl chloridein the presence of a suitable base, such as for example dipotassiumcarbonate and a suitable reaction-inert solvent, such as for examplemethylene chloride.

Compounds of formula (I-B-3) can also be prepared from a compound offormula (I-A), wherein R² is C₁₋₆alkyloxy, said compound beingrepresented by formula (I-A-15), by reaction with a suitable acid, suchas hydrochloric acid, in the presence of a suitable reaction-inertsolvent, such as for example tetrahydrofuran.

Compounds of formula (I-B-3) can be converted into a compound of formula(I-B), wherein R⁵ represents C₁₋₆alkyl, said compound being representedby formula (I-B-4), by reaction with an appropriate alkylating agent,such as for example an intermediate of formula (XIV), wherein W₃represents a suitable leaving group such as a halo atom e.g. iodo, inthe presence of potassium tert. butoxide and in the presence of asuitable reaction-inert solvent, such as for example tetrahydrofuran.

Compounds of formula (I-B-3) can also be converted into a compound offormula (I-B), wherein R⁵ is C₁-alkyloxycarbonylC₁₋₆alkyl orarylC₁₋₆alkyl, said R⁵ being represented by R^(5a) and said compoundbeing represented by formula (I-B-5), by reaction with an intermediateof formula (XV), wherein W₄ represents a suitable leaving group, such asa halo atom, e.g. bromo, chloro and the like, in the presence of asuitable base, such as for example sodium hydride and a suitablereaction-inert solvent, such as for example N,N-dimethylformamide.

Compounds of formula (I-A-2) can also be converted into a compound offormula (I-B), wherein R⁵ is hydrogen and Y is S, said compound beingrepresented by formula (I-B-6), by reaction with H₂N—C(═S)—NH₂ in thepresence of a suitable base, such as potassium hydroxide, and a suitablereaction-inert solvent, such as an alcohol, for example ethanol, orwater. Compounds of formula (I-B-6) can further be converted into acompound of formula (I-A), wherein R² is C₁₋₆alkylthio, said compoundbeing represented by formula (I-A-16), by reaction with a suitableC₁₋₆alkylhalide, such as for example C₁₋₆alkyliodide, in the presence ofa suitable base, such as dipotassium carbonate, and a suitable solvent,such as for example acetone.

Compounds of formula (I_(A/B)-a) can be converted into a compounds offormula (I-A) or (I-B), wherein X is N—R⁷, said compound beingrepresented by formula (I_(A/B)-b), by reaction with an intermediate offormula (XVI), optionally in the presence of a suitable base, such asfor example N,N-diethylethanamine, and in the presence of a suitablereaction-inert solvent, such as an alcohol, e.g. ethanol.

As already indicated in the preparation procedure of compounds offormula (I-A-13) described above, the compounds of formula (I) may alsobe converted to the corresponding N-oxide forms following art-knownprocedures for converting a trivalent nitrogen into its N-oxide form.Said N-oxidation reaction may generally be carried out by reacting thestarting material of formula (I) with an appropriate organic orinorganic peroxide. Appropriate inorganic peroxides comprise, forexample, hydrogen peroxide, alkali metal or earth alkaline metalperoxides, e.g. sodium peroxide, potassium peroxide; appropriate organicperoxides may comprise peroxy acids such as, for example,benzenecarboperoxoic acid or halo substituted benzenecarboperoxoic acid,e.g. 3-chlorobenzenecarboperoxoic acid, peroxoalkanoic acids, e.g.peroxoacetic acid, alkylhydroperoxides, e.g. tert-butyl hydroperoxide.Suitable solvents are, for example, water, lower alkanols, e.g. ethanoland the like, hydrocarbons, e.g. toluene, ketones, e.g. 2-butanone,halogenated hydrocarbons, e.g. dichloromethane, and mixtures of suchsolvents.

Some of the intermediates and starting materials used in the abovereaction procedures are commercially available, or may be synthesizedaccording to procedures already described in the literature.

Intermediates of formula (II) may be prepared by reacting anintermediate of formula (XVII) with an intermediate of formula (XVIII),wherein W₅ represents a suitable leaving group such as a halo atom, e.g.chloro, bromo and the like, in the presence of magnesium, diethyletherand a suitable reaction-inert solvent, such as diethylether.

Intermediates of formula (XVII) may be prepared by oxidizing anintermediate of formula (XIX) in the presence of a suitable oxidizingagent, such as MnO₂, and a suitable reaction-inert solvent, such asmethylene chloride.

Intermediates of formula (XIX) can be prepared by reducing anintermediate of formula (XX) in the presence of a suitable reducingagent such as lithium aluminium hydride, and a suitable reaction-inertsolvent, such as tetrahydrofuran.

Intermediates of formula (XX), wherein Q represents a quinoline moietyoptionally substituted in position 3 with C₁₋₆alkyl and wherein thecarbonyl moiety is placed in position 6, said intermediates beingrepresented by formula (XX-a), can be prepared by reacting anintermediate of formula (XXI) with an intermediate of formula (XXII) inthe presence of sodium 3-nitro-benzene sulfonate, a suitable acid, suchas sulfuric acid, and a suitable alcohol, e.g. methanol, ethanol,propanol, butanol and the like.

Alternatively, intermediates of formula (II) can also be prepared byreacting an intermediate of formula (XXIII) with an intermediate offormula (XXIV), wherein W₆ is a suitable leaving group, such as a haloatom, e.g. bromo, chloro and the like, in the presence of a suitableagent, such as butyl lithium and a suitable reaction-inert solvent, suchas tetrahydrofuran.

Intermediates of formula (XXIII) can be prepared by oxidizing anintermediate of formula (XXV) using the Moffatt Pfitzner or Swernoxidation (dimethylsulfoxide adducts with dehydrating agents e.g. DCC,Ac₂O, SO₃, P₄O₁₀, COCl₂ or Cl—CO—COCl) in an inert solvent such asmethylene chloride.

Intermediates of formula (XXV) can be prepared by reducing anintermediate of formula (XXVI) in the presence of a suitable reducingagent, such as for example lithium aluminium hydride and a suitablereaction-inert solvent, such as benzene.

Intermediates of formula (XXVI) can be prepared from an intermediate offormula (XXVII) by esterification in the presence of a suitable alcohol,such as methanol, ethanol, propanol, butanol and he like, and a suitableacid, such as sulfuric acid.

Intermediates of formula (XXVII), wherein R¹ represents a radical offormula (a-1) with Z, being O, Z₂ being CH₂ and n being 1, saidintermediates being represented by formula (XXVII-a), can be prepared byreducing an intermediate of formula (XXVIII) in the presence of asuitable reducing agent such as hydrogen, and a suitable catalyst, suchas palladium on charcoal, and a suitable acid such as acetic acid. WhenR¹ of intermediate (XXVII) represents an optionally substituted phenylmoiety, it can also be converted into an optionally substitutedcyclohexyl moiety by reduction in the presence of a suitable reducingagent such as rhodium on Al₂O₃, and a suitable reaction-inert solvent,such as tetrahydrofuran.

Intermediates of formula (IV), wherein Q represents a quinoline moietysubstituted in position 2 with halo, e.g. chloro, said intermediatesbeing represented by formula (IV-a), can be prepared by reacting anintermediate of formula (IV), wherein Q represents a quinolinone moietywith R⁵ being hydrogen, said intermediate being represented by formula(IV-b), in the presence of POCl₃.

Intermediates of formula (IV-a), wherein R⁴ is hydrogen, saidintermediates being represented by formula (IV-a-1), can also beprepared by reacting an intermediate of formula (XXIX) with POCl₃ in thepresence of N,N-dimethylformamide (Vilsmeier-Haack formylation followedby cyclization).

Intermediates of formula (XXIX) may be prepared by reacting anintermediate of formula (XXX) with an intermediate of formula (XXXI),wherein W₇ represents a suitable leaving group, such as a halo atom,e.g. chloro, in the presence of a suitable base, such as for exampleN,N-diethylethanamine, and a suitable reaction-inert solvent, such asmethylene chloride.

Intermediates of formula (IV-a) can be converted into an intermediate offormula (IV-c) by reaction with an intermediate of formula (XXXII) inthe presence of a suitable reaction-inert solvent, such as an alcohol,e.g. methanol and the like.

Intermediates of formula (IV-a) can also be converted into anintermediate of formula (IV-d-1) by reaction with a suitable amine offormula (XXXIII-a), wherein Z₃ and Z₄ each independently representhydrogen, C₁₋₆alkyl, C₁₋₆alkyloxyC₁₋₆alkyl, C₁₋₆alkylthioC₁₋₆alkyl orinto an intermediate of formula (IV-d-2) by reaction with a suitableamine of formula (XXXIII-b), wherein Z₃ and Z₄ are taken together toform a heterocycle as defined hereinabove in the definition of R²provided that the heterocycle comprises at least one nitrogen atom, inthe presence of a suitable base, such as for example dipotassiumcarbonate, and a reaction-inert solvent, such as N,N-dimethylformamide.

Intermediates of formula (IV-a), wherein R³ represents CH₂—CH₂—CH₂—Cl,said intermediates being represented by formula (IV-a-2), can also beconverted into an intermediate of formula (IV), wherein R² and R³ aretaken together to form a bivalent radical of formula —O—CH₂—CH₂—CH₂—,said intermediate being represented by formula (IV-e-1), by reactionwith a suitable acid, such as hydrochloric acid and the like.

Intermediates of formula (IV-a-2) can also be converted into anintermediate of formula (IV), wherein R² and R³ are taken together toform a bivalent radical of formula —S—CH₂—CH₂—CH₂—, said intermediatebeing represented by formula (IV-e-2), by reaction with H₂N—C(═S)—NH₂ inthe presence of a suitable reaction-inert solvent, such as an alcohol,e.g. ethanol.

Intermediates of formula (V) may be prepared by reacting an intermediateof formula (XXVII) with an intermediate of formula CH₃—NH—O—CH₃ in thepresence of 1,1′-carbonyldiimidazole and a suitable reaction-inertsolvent, such as methylene chloride.

Intermediates of formula (VII), wherein Q represents a quinoline moiety,in particular a quinoline moiety wherein R² is ethyl, R³ is methyl andR⁴ is hydrogen, and the carboxyl moiety is placed in position 6, saidintermediates being represented by formula (VII-a), can be prepared byreaction an intermediate of formula (XXXIV) in the presence of asuitable aldehyde, such as CH₃—CH₂—CH(═O), (CH₂O)_(n), ZnCl₂, FeCl₃ anda suitable reaction-inert solvent, such as an alcohol, for exampleethanol.

Intermediates of formula (VIII) can be prepared by reacting anintermediate of formula (XXXV) with an intermediate of formula (XXXVI)in the presence of a suitable catalyst, such as for exampletetrakis(triphenylphosphine)palladium and a suitable reaction-inertsolvent, such as for example dioxane.

Still some other preparations can be devised, some of them are disclosedfurther in this application with the Examples.

Pure stereoisomeric forms of the compounds and the intermediates of thisinvention may be obtained by the application of art-known procedures.Diastereomers may be separated by physical separation methods such asselective crystallization and chromatographic techniques, e.g. liquidchromatography using chiral stationary phases. Enantiomers may beseparated from each other by the selective crystallization of theirdiastereomeric salts with optically active acids. Alternatively,enantiomers may be separated by chromato-graphic techniques using chiralstationary phases. Said pure stereoisomeric forms may also be derivedfrom the corresponding pure stereoisomeric forms of the appropriatestarting materials, provided that the reaction occurs stereo-selectivelyor stereospecifically. Preferably, if a specific stereoisomer isdesired, said compound will be synthesized by stereoselective orstereospecific methods of preparation. These methods will advantageouslyemploy chirally pure starting materials. Stereoisomeric forms of thecompounds of formula (I) are obviously intended to be included withinthe scope of the invention.

A stereoisomer of a compound of formula (I-A) or (I-B) such as a cisform, may be converted into another stereoisomer such as thecorresponding trans form by reacting the compound with a suitable acid,such as hydrochloric acid, in the presence of a suitable reaction-inertsolvent, such as for example tetrahydrofuran.

The mGluR1 antagonistic activity of the present compounds can bedemonstrated in the Signal transduction on cloned rat mGluR1 in CHOcells test and the Cold allodynia test in rats with a Bennett ligation,as described hereinafter.

Due to their mGluR antagonistic activity, more in particular their GroupI mGluR antagonistic activity and even more in particular, their mGluR1antagonistic activity, the compounds of formula (I-A) or (I-B), theirN-oxides, pharmaceutically acceptable addition salts, quaternary aminesand stereochemically isomeric forms are useful in the treatment orprevention of glutamate-induced diseases of the central nervous sytem.Diseases in which a role for glutamate has been demonstrated includedrug addiction or abstinence (dependence, opioid tolerance, opioidwithdrawal), hypoxic, anoxic and ischemic injuries (ischemic stroke,cardiac arrest), pain (neuropathic pain, inflammatory pain,hyperalgesia), hypoglycemia, diseases related to neuronal damage, braintrauma, head trauma, spinal cord injury, myelopathy, dementia, anxiety,schizophrenia, depression, impaired cognition, amnesia, bipolardisorders, conduct disorders, Alzheimer's disease, vascular dementia,mixed (Alzheimer's and vascular) dementia, Lewy Body disease, deliriumor confusion, Parkinson's disease, Huntington's disease, Down syndrome,epilepsy, aging, Amyotrophic Lateral Sclerosis, multiple sclerosis, AIDS(Acquired Immune Deficiency Syndrome) and AIDS related complex (ARC).

The present invention also provides compositions for the administrationto mamals, in particular humans, in particular for diagnostic reasons,more in particular for imaging an organ comprising a therapeuticallyeffective amount of a radiolabelled compound of formula (I-A)* or (I-B)*and a pharmaceutically acceptable carrier or diluent.

Therefore, the compounds of the present invention or any subgroupthereof may be formulated into various pharmaceutical forms foradministration purposes. As appropriate compositions there may be citedall compositions usually employed for systemically administering drugs.To prepare the pharmaceutical compositions of this invention, atherapeutically effective amount of a particular compound, in base oraddition salt form, as the active ingredient is combined in intimateadmixture with a pharmaceutically acceptable carrier, which carrier maytake a wide variety of forms depending on the form of preparationdesired for administration. These pharmaceutical compositions aredesirably in unitary dosage form suitable, preferably, foradministration orally, rectally, topically, percutaneously or byparenteral injection. For example, in preparing the compositions in oraldosage form, any of the usual pharmaceutical media may be employed, suchas, for example, water, glycols, oils, alcohols and the like in the caseof oral liquid preparations such as suspensions, syrups, emulsions,elixirs and solutions: or solid carriers such as starches, sugars,kaolin, lubricants, binders, disintegrating agents and the like in thecase of powders, pills, capsules and tablets. Because of their ease inadministration, tablets and capsules represent the most advantageousoral dosage unit form, in which case solid pharmaceutical carriers areobviously employed. For parenteral compositions, the carrier willusually comprise sterile water, at least in large part, though otheringredients, for example, to aid solubility, may be included. Injectablesolutions, for example, may be prepared in which the carrier comprisessaline solution, glucose solution or a mixture of saline and glucosesolution. Injectable suspensions may also be prepared in which caseappropriate liquid carriers, suspending agents and the like may beemployed. Also included are solid form preparations which are intendedto be converted, shortly before use, to liquid form preparations. Asappropriate compositions for topical application there may be cited allcompositions usually employed for topically administering drugs e.g.creams, gel, dressings, shampoos, tinctures, pastes, ointments, salves,powders and the like. In the compositions suitable for percutaneousadministration, the carrier optionally comprises a penetration enhancingagent and/or a suitable wetting agent, optionally combined with suitableadditives of any nature in minor proportions, which additives do notcause a significant deleterious effect to the skin. Said additives mayfacilitate the administration to the skin and/or may be helpful forpreparing the desired compositions. These compositions may beadministered in various ways, e.g., as a transdermal patch, as aspot-on, as an ointment.

It is especially advantageous to formulate the aforementionedpharmaceutical compositions in unit dosage form for ease ofadministration and uniformity of dosage. Unit dosage form as used in thespecification and claims herein refers to physically discrete unitssuitable as unitary dosages, each unit containing a predeterminedquantity of active ingredient calculated to produce the desiredtherapeutic effect in association with the required pharmaceuticalcarrier. Examples of such unit dosage forms are tablets (includingscored or coated tablets), capsules, pills, suppositories, powderpackets, wafers, injectable solutions or suspensions, teaspoonfuls,tablespoonfuls and the like, and segregated multiples thereof.

The diagnostically effective dose or frequency of administration dependson the particular compound of formula (I-A)* or (I-B)* used and theparticular condition of the mamal being treated, as is well known tothose skilled in the art.

The following examples are intended to illustrate and not to limit thescope of the present invention.

Experimental Part

Hereinafter, “DMF” is defined as N,N-dimethylformamide, “DIPE” isdefined as diisopropylether, “DMSO” is defined as dimethylsulfoxide,“BHT” is defined as 2,6-bis(1,1-dimethylethyl)-4-methylphenol, and “THF”is defined as tetrahydrofuran.

A. Preparation of the Intermediates

EXAMPLE A1 Preparation of

A mixture of 4-(1-methylethoxy)benzoic acid (0.083 mol) and Rh/Al₂O₃ 5%(log) in THF (220 ml) was hydrogenated at 50° C. (under 3 bar pressureof H₂) for 1 night. The mixture was filtered over celite, washed withTHF and evaporated. Yield: 16 g of intermediate 1 (100%).

EXAMPLE A2 Preparation of 2-ethyl-3-methyl-6-quinolinecarboxylic acid(interm. 2)

A mixture of 4-aminobenzoic acid (0.299 mol) in ethanol (250 ml) wasstirred at room temperature. ZnCl₂ (0.0367 mol) and (CH₂O)_(n) (10 g)were added. FeCl₃.6H₂O (0.5 mol) was added portionwise and thetemperature rised till 60-65° C. Propanal (30 ml) was added dropwiseover a 2 hours period. The mixture was refluxed for 2 hours and kept atroom temperature for 12 hours. The mixture was poured into water andfiltered through celite. The filtrate was acidified till pH=7 with HCl6N and the mixture was evaporated till dryness. The residue was usedwithout further purification. Yield: 56.1 g of2-ethyl-3-methyl-6-quinolinecarboxylic acid (interm. 2).

EXAMPLE A3 Preparation of

Pentanoyl chloride (0.2784 mol) was added at 5° C. to a mixture of4-bromobenzenamine (0.232 mol) and N,N-diethylethanamine (0.2784 mol) inCH₂Cl₂ (400 ml). The mixture was stirred at room temperature overnight,poured out into water and extracted with CH₂Cl₂. The organic layer wasseparated, washed with a concentrated NH₄OH solution and water, dried(MgSO₄), filtered and the solvent was evaporated. The residue (60 g) wascrystallized from diethylether. The precipitate was filtered off anddried. The residue (35 g, 63%) was taken up in CH₂Cl₂. The organic layerwas separated, washed with a 10% K₂CO₃ solution, washed with water,dried (MgSO₄), filtered and the solvent was evaporated. Yield: 30 g ofintermediate (3) (54%).

EXAMPLE A4 Preparation of

A mixture of 6-bromo-2(1H)-quinolinone (0.089 mol) in POCl₃ (55 ml) wasstirred at 60° C. overnight, then at 100° C. for 3 hours and the solventwas evaporated. The residue was taken up in CH₂Cl₂, poured out into icewater, basified with NH₄OH conc., filtered over celite and extractedwith CH₂Cl₂. The organic layer was separated, dried (MgSO₄), filteredand the solvent was evaporated. Yield: 14.5 g of intermediate (4) (67%).

EXAMPLE A5 a) Preparation of

DMF (37 ml) was added dropwise at 10° C. under N₂ flow to POCl₃ (108ml). After complete addition, the mixture was allowed to warm to roomtemperature. N-(4-bromophenyl)butanamide (0.33 mol) was addedportionwise. The mixture was stirred at 85° C. overnight, then allowedto cool to room temperature and poured out on ice (exothermic reaction).The precipitate was filtered off, washed with a small amount of waterand dried (vacuum). The residue was washed with EtOAc/diethyl ether anddried. Yield: 44.2 g of intermediate (5) (50%).

b) Preparation of

A mixture of intermediate (5) (0.162 mol) in methanol (600 ml), and asolution of methanol sodium salt in methanol at 35% (154 ml) was stirredand refluxed overnight. The mixture was poured out on ice. Theprecipitate was filtered off, washed with a small amount of water andtaken up in CH₂Cl₂. K₂CO₃ 10% was added and the mixture was extractedwith CH₂Cl₂. The organic layer was separated, washed with water, dried(MgSO₄), filtered and the solvent was evaporated. Yield: 31.9 g ofintermediate (6) (74%).

EXAMPLE A6 Preparation of

1,1′-Carbonylbis-1H-imidazole (0.074 mol) was added portionwise to amixture of 4-methoxycyclohexanecarboxylic acid (0.063 mol) in CH₂Cl₂(200 ml). The mixture was stirred at room temperature for 1 hour. ThenN-methoxymethanamine (0.074 mol) was added. The mixture was stirred atroom temperature overnight, poured out into H₂O and extracted withCH₂Cl₂. The organic layer was separated, washed several times with H₂O,dried (MgSO₄), filtered and the solvent was evaporated. Yield: 12.6 g ofinterm. 7.

EXAMPLE A7

a) A mixture of 6-fluoro-4-oxo-4H-1-benzopyran-2-carboxylic acid (0.30mol) in acetic acid (400 ml) was hydrogenated with Pd/C (3 g) as acatalyst. After uptake of H₂ (3 equiv), the catalyst was filtered off.The filtrate was evaporated. The residue was stirred in petroleum ether.The precipitate was filtered off and dried (vacuum; 70° C.). Afterrecrystallization from CHCl₃/CH₃OH, the precipitate was filtered off anddried (vacuum; 80° C. and high vacuum; 85° C.). Yield: 8.8 g of6-fluoro-3,4-dihydro-2H-1-benzopyran-2-carboxylic acid (interm. 8)(15.0%).

b) A mixture of intermediate (8) (0.255 mol) in ethanol (400 ml) andH₂SO₄ (5 ml) was stirred and refluxed for 8 hours. The solvent wasevaporated till dryness. The residue was dissolved in CH₂Cl₂. Theorganic layer was separated, washed with K₂CO₃ 10%, dried (MgSO₄),filtered and the solvent was evaporated. Yield: 45 g of ethyl6-fluoro-3,4-dihydro-2H-1-benzopyran-2-carboxylate (interm. 9) (79%).

c) Reaction under N₂. A mixture of sodiumbis(2-methoxyethoxy)aluminumhydride, 70 wt % solution in methylbenzene3.4M (0.44 mol) in benzene (150 ml) (reflux) was added dropwise during 1hour to a refluxed mixture of interm. 9 (0.22 mol) and benzene (600 ml).After stirring for 2.5 hours at reflux temperature, the mixture wascooled to ±15° C. The mixture was decomposed by adding dropwise ethanol(30 ml) and water (10 ml). This mixture was poured out onto ice/waterand this mixture was acidified with concentrated hydrochloric acid. Thismixture was extracted with diethyl ether (500 ml). The separated organiclayer was washed with water, dried, filtered and the solvent wasevaporated. The residue was purified by column chromotoghaphy oversilica gel (eluent: CHCl₃). The desired fraction was collected and theeluent was evaporated. Yield: 34 g of6-fluoro-3,4-dihydro-2H-1-benzopyran-2-methanol (interm. 10) (85%).

d) Reaction under N₂. To a stirred and cooled (−60° C.; 2-propanone/CO₂bath) mixture of ethanedioyl dichloride (0.1 mol) in CH₂Cl₂ (350 ml) wasadded sulfinylbis[methane] (30 ml) during 10 minutes. After stirring 10minutes, a mixture of interm. 10 in CH₂Cl₂ (90 ml) was added during 5minutes. After stirring for 15 minutes, N,N-diethylethanamine (125 ml)was added. When the mixture was warmed up to room temperature, it waspoured out in water. The product was extracted with CH₂Cl₂. The organiclayer was wased with water, HCl (1M), water, NaHCO₃ (10%) and water,dried and evaporated. The residue was dissolved in diethyl ether, washedwith water, dried, filtered and evaporated. The residue was purified bycolumn chromotoghaphy over silica gel (eluent: CHCl₃). The desiredfraction was collected and the eluent was evaporated. Yield: 21.6 g of6-fluoro-3,4-dihydro-2H-1-benzopyran-2-carboxaldehyde (interm. 11)(67%).

e) Preparation of

nButyllithium 1.6M (0.056 mol) was added slowly at −70° C. to a solutionof intermediate (5) (0.046 mol) in THF (100 ml). The mixture was stirredat −70° C. for 30 minutes. A suspension of interm. 11 (0.056 mol) in THF(100 ml) was added slowly. The mixture was stirred at −70° C. for 1hour, then brought to room temperature, poured out into H₂O andextracted with EtOAc. The organic layer was separated, dried (MgSO₄),filtered and the solvent was evaporated. The residue (21 g) was purifiedby column chromatography over silica gel (eluent: cyclohexane/EtOAc80/10; 15-35 μm). The pure fractions were collected and the solvent wasevaporated. Yield: 9.5 g of interm. 12 (55%).

EXAMPLE A8 a) Preparation of

A mixture of intermediate (5) (0.1127 mol), 2-methoxyethanamine (0.2254mol) and K₂CO₃ (0.2254 mol) in DMF (500 ml) was stirred at 120° C. for15 hours and then cooled. The solvent was evaporated. The residue wastaken up in CH₂Cl₂ and H₂O. The organic layer was separated, dried(MgSO₄), filtered and the solvent was evaporated till dryness. Theresidue (33.53 g) was purified by column chromatography over silica gel(eluent: CH₂Cl₂/CH₃OH 99.5/0.5; 15-40 μm). Two fractions were collectedand their solvents were evaporated. Yield: 5.7 g of interm. 14 (38%) andinterm. 13 (34%).

b) Preparation of

A mixture of intermediate (5) (0.0751 mol), thiomorpholine (0.0891 mol)and K₂CO₃ (0.15 mol) in DMF (200 ml) was stirred at 120° C. for 12hours. The solvent was evaporated till dryness. The residue was taken upin CH₂Cl₂ and H₂O. The organic layer was separated, dried (MgSO₄),filtered and the solvent was evaporated. The residue (26 g) was purifiedby column chromatography over silica gel (eluent: cyclohexane/EtOAc80/20; 20-45 μm). Two fractions were collected and their solvents wereevaporated. The two fractions were combined. Yield: 9.4 g of interm. 15(37%); mp. 82° C.

EXAMPLE A9

a) 4-Aminobenzoic acid (0.219 mol) was added to a solution of sodium3-nitrobenzenesulfonate (0.118 mol) in H₂SO₄ 70% (230 ml) and themixture was stirred and refluxed. 2-propene-1,1-diol, 2-methyl-,diacetate (0.216 mol) was added dropwise and the mixture was refluxedfor 4 hours. Ethanol (200 ml) was added and the mixture was stirred at80° C. for 48 hours. The mixture was evaporated, the residue was pouredinto ice water/NH₄OH and extracted with CH₂Cl₂. The organic layer wasdried (MgSO₄) and evaporated. The residue was purified by columnchromatography over silica gel (eluent: CH₂Cl₂/2-propanol 99/1). Thepure fractions were collected and evaporated. Yield: 21 g of ethyl3-methyl-6-quinolinecarboxylate (interm. 16) (45%).

b) Interm. 16 (0.098 mol) in THF (270 ml) was added at 0° C. to asolution of LiAlH₄ (0.098 mol) in THF under N₂. When the addition wascomplete, water (10 ml) was added. The precipitate was filtered off andwashed with CH₂Cl₂. The organic layer was dried (MgSO₄), filtered offand evaporated. The product was used without further purification.Yield: 16.71 g of 3-methyl-6-quinolinemethanol (interm. 17).

c) MnO₂ (0.237 mol) was added to a solution of interm. 17 (0.096 mol) inCH₂Cl₂ (200 ml) and the mixture was stirred at room temperature for 12hours. The mixture was filtered through celite and the filtrate wasstirred again with MnO₂ (20 g) for 12 hours. MnO₂ (10 g) was addedagain. The mixture was stirred for 12 hours. The mixture was filteredthrough celite and evaporated. The product was used without furtherpurification. Yield: 11.71 g of 3-methyl-6-quinolinecarboxaldehyde (71%)(interm. 18).

d) A solution of bromocyclohexyl (0.14 mol) in 1,1′-oxybisethane (50 ml)and Mg turnings (50 ml) was added at 10° C. to a mixture of THF (0.14mol) in 1,1′-oxybisethane (10 ml). A solution of interm. 18 (0.07 mol)in Mg turnings (100 ml) was added carefully at 5° C., the mixture waspoured into ice water and extracted with EtOAc. Yield: 11.34 g of(±)-α-cyclohexyl-3-methyl-6-quinolinemethanol (63%) (interm. 19).

EXAMPLE A10 Preparation of

A mixture of compound (5) (0.001507 mol),tributyl(1-ethoxyethenyl)stannane (0.00226 mol) and Pd(PPh₃)₄ (0.000151mol) in 1,4-dioxane (5 ml) was stirred at 80° C. for 3 hours. Water wasadded. The mixture was extracted with EtOAc. The organic layer wasseparated, dried (MgSO₄), filtered and the solvent was evaporated. Thisproduct was used without further purification. Yield: 1.4 g of interm.20.

EXAMPLE A11 Preparation of

A mixture of compound (45) (prepared according to B6) (0.00125 mol) inNaOH 3N (5 ml) and iPrOH (1.7 ml) was stirred at room temperatureovernight, then poured out into H₂O, acidified with HCl 3N and extractedwith EtOAc. The organic layer was separated, dried (MgSO₄), filtered andthe solvent was evaporated. The residue was taken up in diethyl ether.The precipitate was filtered off and dried. Yielding: 0.26 g ofintermediate 21 (56%). (mp.: 232° C.)

EXAMPLE A12 a. Preparation of

A mixture of 5-bromo-1H-indole-2,3-dione (0.221 mol) in NaOH 3N (500 ml0was stirred at 80° C. for 30 minutes, brought to room temperature and2-pentanone (0.221 mol) was added. The mixture was stirred and refluxedfor 1 hour and 30 minutes and acidified with AcOH until pH=5. Theprecipitate was filtered, washed with water and dried. Yielding 52.3 gof intermediate 22 and intermediate 23. (Total yielding: 80%).

b. Preparation of

nBuLi 1.6 M (0.0816 mol) was added dropwise at −78° C. to a suspensionof intermediate 22 (0.034 mol) and intermediate 23 (0.034 mol) in THF(300 ml) under N₂ flow. The mixture was stirred at −78° C. for 30minutes. nBuLi 1.6M (0.0816 mol) was added dropwise. The mixture wasstirred for 1 hour. A mixture of intermediate 9 (0.102 mol) in THF (250ml) was added slowly. The mixture was stirred for −78° C. to −20° C.,poured out into H₂O/HCl 3N and extracted with EtOAc. The organic layerwas separated, dired (MgSO₄), filtered, and the solvent was evaporatedtill dryness. Yielding: 20.89 g of compound intermediate 24 andintermediate 25 (86%).

EXAMPLE A13 a. Preparation of

4-amino-3-methoxybenzoic acid (0.054 mol) was added portionwise at roomtemperature to a solution of 3-chloro-2-ethyl-2-butenal (0.065 mol) inAcOH (100 ml). The mixture was stirred and refluxed for 8 hours andevaporated to dryness. The residue was taken up in CH₂Cl₂, water wasadded and the solution was basified by Et₃N. The organic layer wasseparated, dried (MgSO₄), filtered, and the solvent was evaporated. Theresidue was crystallized from 2-propanone. The precipitate was filteredoff and dried. Yielding: 2.5 g of interm. 26 (18%).

b. Preparation of

CDI (0.012 mol) was added at room temperature to a solution of interm.26 (0.011 mol) in CH₂Cl₂ (30 ml). The mixture was stirred at roomtemperature for 1 hour. methoxyaminomethyl (0.012 mol) was added and themixture was stirred at room temperature for 8 hours. H₂O was added. Aprecipitate was filtered off. The filtrate was extracted with CH₂Cl₂.The organic layer was separated, dried (MgSO₄), filtered, and thesolvent was evaporated. The residue was crystallized from diethyl ether.The precipitate was filtered off and dried. Yielding: 0.95 g of interm.27 (31%) (mp.: 148° C.).

EXAMPLE A14 Preparation of

4-Bromobenzenamine (0.034 mol) was added at room temperature to asolution of 3-chloride-2-ethyl-2-butanal (0.041 mol) in AcOH (60 ml).The mixture was stirred and refluxed for 8 hours, brought to roomtemperature and evaporated to dryness. The product was crystallized fromEtOAc. The precipitate was filtered, washed with K₂CO₃ 10% and taken upin CH₂Cl₂. The organic layer was separated, dried (MgSO₄), filtered, andthe solvent was evaporated. Yielding: 4.6 g of interm. 28 (54%).

EXAMPLE A15 a. Preparation of

A solution of KOH (0.326 mol) in H₂O (150 ml) was added slowly at 5° C.to a solution of 1,3-cyclohexanedione (0.268 mol) in H₂O (150 ml). Thetemperature must not reach 12° C. KI (2 g) then2-bromo-1-(4-nitrophenyl)ethanone (0.294 mol) were added portionwise.The mixture was stirred at room temperature for 48 hours. Theprecipitate was fitered, washed with H₂O then with diethyl ether anddried. Yielding: 63 g (85%). A part of this fraction (1 g) wascrystallized from EtOH. The precipitate was filtered off and dried.Yielding: 0.5 g of interm. 29 (42%) (mp.: 100° C.).

b. Preparation of

A mixture of interm. 29 (0.145 mol) in H₂SO₄ (40 ml) was stirred at roomtemperature for 1 hour, poured out into ice, basified with NH₄OH andextracted with CH₂Cl₂. The organic layer was separated, dried (MgSO₄),filtered, and the solvent was evaporated. The residue was crystallizedfrom EtOH. The precipitate was filtered off and dried. Yielding: 31 g(58%). A part of this fraction (1 g) was crystallized from EtOH. Theprecipitate was filtered off and dried. Yielding: 0.7 g of interm. 30(58%) (mp.: 200° C.).

c. Preparation of

A mixture of interm. 30 (0.039 mol), Raney Ni (10 g) in EtOH (100 ml)was hydrogenated at room temperature under a 3 bar pressure for 1 hour.The mixture was filtered over celite and washed with CH₂Cl₂. The organiclayer was separated, dried (MgSO₄), filtered, and the solvent wasevaporated. The residue (9.5 g) was crystallized from diethyl ether. Theprecipitate was filtered off and dried. Yielding: 4.6 g (52%). Thefiltrate was evaporated. The residue (2.7 g) was purified by columnchromatography over silica gel (eluent: CH₂Cl₂/CH₃OH; 99/1; 15-40 μm).Two fractions were collected and the solvent was evaporated. Yielding:1.6 g F1 and 1.2 g F2. F2 was crystallized from EtOH. The precipitatewas filtered off and dried. Yielding: 0.24 g of interm. 31 (2%) (mp.:202° C.).

c. Preparation of

Interm. 30 (0.02 mol) was added at room temperature to a solution of3-chloro-2-ethyl-2-butenal (0.04 mol) in AcOH (50 ml). The mixture wasstirred and refluxed for 4 hours. The solvent was evaporated tilldryness. The residue was crystallized from EtOAc. The precipitate wasfiltered off and dried. The residue was taken up in CH₂Cl₂. The mixturewas basified with K₂CO₃ 10% and extracted with CH₂Cl₂. The organic layerwas separated, dried (MgSO₄), filtered, and the solvent was evaporated.The residue was crystallized from EtOH. The precipitate was filtered offand dried. Yielding: 2.5 g of interm. 32 (40%).

EXAMPLE A16 Preparation of

A mixture of 2-(4-nitrophenyl)-1-phenylethanone (0.083 mol) and Raney Ni(20 g) in EtOH (200 ml) was hydrogenated at room temperature for 1 hourunder a 3 bar pressure, then filtered over celite, washed withCH₂Cl₂/CH₃OH and dried. Yielding: 17.5 g of interm. 33 (97%).

EXAMPLE A17 a. Preparation of

DMF (12.4 ml) was added dropwise at 5° C. to POCl₃ (0.7536 mol).4′-bromo-5-chlorovaleranilide (0.1032 mol) was added and the mixture wasstirred at 75° C. for 6 hours, cooled at room temperature and poured outinto ice water. The insoluble was filtered, washed with water and dried.Yielding: 25.7 g of intermediate 34 (78%).

b. Preparation of

A mixture of intermediate 34 (0.094 mol) in HCl 6N (250 ml) was stirredand refluxed for 2 days, cooled, poured out on water (100 ml) andneutralyzed with NH₄OH (concentrated). The insoluble was filtered andwashed with water then with EtOH. Yielding: 19 g. The filtrate wasevaporated. The residue (9.4 g) was purified by column chromatographyover silica gel (eluent: CH₂Cl₂/CH₃OH 99.25/0.75; 15-35 μm). Onefraction was collected and the solvent was evaporated. Yielding: 8 g ofintermediate 35 (32%).

B. Preparation of the Non-Radioactive Compounds

EXAMPLE B1 Preparation of

POCl₃ (0.024 mol) was added slowly at 5° C. to DMF (0.024 mol). Themixture was stirred at room temperature for 30 minutes, then cooled to5° C. 3-Oxo-butanoic acid ethyl ester (0.024 mol) was added slowly. Themixture was stirred at 5° C. for 30 minutes.1-(4-aminophenyl)-2-phenylethanone (0.024 mol) was added portionwise.The mixture was stirred at 90° C. for 3 hours and dissolved in CH₂Cl₂.Ice water was added. The mixture was basified with NH₄OH and extractedwith CH₂Cl₂. The organic layer was separated, dried (MgSO₄), filtered,and the solvent was evaporated. The residue was crystallized from2-propanone/diethyl ether. The precipitate was filtered off and dried.Yielding: 0.9 g of compound 306 (11%) (mp.: 136° C.).

EXAMPLE B2 Preparation of

KMnO₄ (10 g) was added portionwise at room temperature to a solution of

(prepared according to example A7.e) (0.022 mol) intris(dioxa-3,6-heptyl)amine (1 ml) and CH₂Cl₂ (100 ml). The mixture wasstirred at room temperature for 8 hours, filtered over celite, washedwith CH₂Cl₂ and dried. The residue (6 g, 100%) was crystallized fromdiethyl ether/petroleum ether. The precipitate was filtered off anddried. Yield: 2 g of compound (2) (33%); mp. 82° C.

EXAMPLE B3 a) Preparation of

nBuLi 1.6M (0.07 mol) was added slowly at −70° C. to a solution ofintermediate (5) (0.058 mol) in THF (150 ml). The mixture was stirred at−70° C. for 30 minutes. A solution of2,3-dihydro-1H-Indene-2-carbonitrile (0.07 mol) in THF (100 ml) wasadded slowly. The mixture was stirred at −70° C. for 1 hour, broughtslowly to room temperature, hydrolized with H₂O and extracted withEtOAc. The organic layer was separated, dried (MgSO₄), filtered and thesolvent was evaporated. The residue (22 g) was purified by columnchromatography over silica gel (eluent: CH₂Cl₂/cyclohexane 80/20 to 100;15-35 μm). The pure fractions were collected and the solvent wasevaporated. The second fraction was crystallized from2-propanone/diethyl ether. The precipitate was filtered off and dried.Yield: 0.11 g of compound (3). The filtrate was concentrated. Yield:0.55 g of compound (3); mp. 145° C.

b) Preparation of

nBuLi 1.6M (0.022 mol) was added slowly at −70° C. to a solution ofintermediate (5) (0.018 mol) in THF (50 ml). The mixture was stirred at−70° C. for 1 hour, brought to −40° C., then cooled to −70° C. Asolution of interm. 7 (0.018 mol) in THF (40 ml) was added slowly. Themixture was stirred at −70° C. for 1 hour, then brought to −20° C.,hydrolyzed with H₂O and extracted with EtOAc. The organic layer wasseparated, dried (MgSO₄), filtered and the solvent was evaporated. Theresidue (6.5 g) was purified by column chromatography over silica gel(eluent: toluene/EtOAc 90/10; 15-40 μM). Two fractions (F1 and F2) werecollected and the solvent was evaporated. F1 (2.4 g) was crystallizedfrom diethyl ether. The precipitate was filtered off and dried. Yield:1.8 g of compound (4) (29%); mp. 123° C. F2 (0.9 g) was crystallizedfrom diethyl ether. The precipitate was filtered off and dried. Yield:0.2 g of compound (5) (3%); mp. 120° C.

c) Preparation of

nBuLi 1.6M in exane (0.107 mol) was added dropwise at −78° C. under N₂flow to a mixture of intermediate (6) (0.089 mol) in THF. The mixture wsstirred at −78° C. for 1 hour. A mixture of interm. 7 (150 ml) was addedat −78° C. under N₂ flow. The mixture was stirred at −78° C. for 2hours, brought to 0° C., poured out into H₂O and extracted with EtOAc.The organic layer was separated, dried (MgSO₄), filtered and the solventwas evaporated. The residue (31 g) was purified by column chromatographyover silica gel (eluent: cyclohexane/EtOAc 85/15; 20-45 μm). Two purefractions were collected and their solvents were evaporated. Yielding:11 g of compound (7) (38%) and 8.2 g of compound (8) (28%).

d) Preparation of

A solution of chloromethylbenzeen (0.0069 mol) in diethyl ether (8 ml)was added slowly to a suspension of Mg (0.0069 mol) in a small amount ofdiethyl ether. The mixture was stirred at room temperature for 30minutes (disparition of Mg), then cooled to 5° C. A solution of interm.27 (0.0027 mol) in THF (8 ml) was added slowly. The mixture was stirredat 5° C. for 15 minutes, then at room temperature for 2 hours, pouredout into H₂O and filtered over celite. The precipitate was washed withEtOAc. The filtrate was extracted with EtOAc. The organic layer wasseparated, dried (MgSO₄), filtered, and the solvent was evaporated. Theresidue (1 g) was purified by column chromatography over kromasil(eluent: CH₁—Cl₂ 100 to CH₂Cl₂/CH₃OH 99/1; 15-40 μm). The pure fractionswere collected and the solvent was evaporated. The residue (0.5 g, 56%)was crystallized from diethyl ether. The precipitate was filtered offand dried. Yielding: 0.14 g of compound 503 (15%).

EXAMPLE B4 EXAMPLES OF ENDGROUP MODIFICATIONS a) Preparation of

A mixture of

(prepared according to example B3.c) (0.018 mol) in HCl 3N (60 ml) andTHF (60 ml) was stirred at 60° C. overnight. The mixture was basifiedwith a K₂CO₃ 10% solution and extracted with CH₂Cl₂. The organic layerwas separated, dried (MgSO₄), filtered and the solvent was evaporated.Yield: 4.6 g of compound (156) (82%).

b) Preparation of

A mixture of

(prepared according to example B3.c) (0.0122 mol) in HCl 3N (40 ml) andTHF (40 ml) was stirred and refluxed overnight, poured out into water,basified with K₂CO₃ 10% and extracted with CH₂Cl₂. The organic layer wasseparated, dried (MgSO₄), filtered and the solvent was evaporated. Theresidue was purified by column chromatography over silica gel (eluent:cyclohexane/EtOAc 40/60; 15-40 μm). The pure fractions were collectedand the solvent was evaporated. Yield: 2 g of compound (9) (52%); mp.226° C.

c) Preparation of

A mixture of compound (4) (0.0015 mol), 2-methoxyethanamine (0.003 mol)and K₂CO₃ (0.003 mol) in DMF (5 ml) was stirred at 140° C. for 48 hours.H₂O was added. The mixture was extracted with EtOAc. The organic layerwas separated, dried (MgSO₄), filtered and the solvent was evaporated.The residue (1 g) was purified by column chromatography over silica gel(eluent: cyclohexane/EtOAc 60/40; 15-40 μm). Two fractions werecollected and the solvent was evaporated. Both fractions werecrystallized separately from pentane. The precipitate was filtered offand dried. Yield: 0.05 g of compound (10) (9%; mp. 115° C.) and 0.057 gof compound (11) (10%; mp. 107° C.).

d) Preparation of

A mixture of compound (4) (0.0015 mol) in 2-(methylthio)ethanamine (2ml) was stirred at 120° C. for 8 hours. K₂CO₃ 10% was added. The mixturewas extracted with CH₂Cl₂. The organic layer was separated, dried(MgSO₄), filtered and the solvent was evaporated. The residue (2.2 g)was purified by column chromatography over silica gel (eluent:cyclohexane/EtOAc 70/30; 15-40 cm). Two fractions were collected and thesolvent was evaporated. The first fraction was crystallized from diethylether/petroleum ether. The precipitate was filtered off and dried.Yield: 0.08 g of compound (12) (14%); mp. 120° C. The second fractionwas crystallized from diethyl ether. The precipitate was filtered offand dried. Yield: 0.18 g of compound (13) (31%); mp. 125° C.,

e) Preparation of

A mixture of compound (4) (0.001507 mol), ethynyltrimethylsilane(0.003013 mol), CuI (0.000151 mol) and Pd(PPh₃)₄ (0.000151 mol) inN,N-diethylethanamine (5 ml) was stirred at 100° C. for 24 hours. Waterwas added. The mixture was filtered over celite, washed with EtOAc andthe filtrate was extracted with EtOAc. The organic layer was separated,dried (MgSO₄), filtered and the solvent was evaporated. The residue (1.3g) was purified by column chromatography over silica gel (eluent:cyclohexane/EtOAc 85/15; 15-40 μm). The pure fractions were collectedand the solvent was evaporated. The residue (0.3 g) was crystallizedfrom pentane. The precipitate was filtered off and dried. Yield: 0.11 gof compound (14) (18%); mp. 114° C.

f) Preparation of

A mixture of compound (14) (0.013 mol) and KF (0.038 mol) in acetic acid(50 ml) was stirred at room temperature for 2 hours. H₂O was added andthe mixture was extracted with diethyl ether. The organic layer wasseparated, dried (MgSO₄), filtered and the solvent was evaporated. Theresidue (4.4 g) was purified by column chromatography over silica gel(eluent: cyclohexane/EtOAc 70/30; 15-40 μm). One fraction was collectedand the solvent was evaporated. This fraction (3 g, 73%) wascrystallized from diethyl ether. The precipitate was filtered off anddried. Yield: 2.45 g of compound (15) (60%); mp. 132° C.

g) Preparation of

A mixture of

prepared according to example B.7.a) (0.0056 mol) in KOH [1M, H₂O] (10ml) and methanol (30 ml) was stirred at room temperature for 1 hour,poured out into water and extracted with EtOAc. The organic layer wasseparated, dried (MgSO₄), filtered and the solvent was evaporated. Theresidue (2.2 g) was purified by column chromatography over silica gel(eluent: cyclohexane/EtOAc 85/15 to 70/30; 15-40 μm). Two fractions werecollected and the solvent was evaporated. The first fraction wascrystallized from diethyl ether. The precipitate was filtered off anddried. Yield: 0.2 g of compound (15) (11%); mp. 133° C. The secondfraction was crystallized from diethyl ether. The precipitate wasfiltered off and dried. Yield: 0.3 g of compound (17) (16%); mp. 128° C.

h) Preparation of

A mixture of compound (4) (0.001205 mol), 2-propyn-1-ol (0.002411 mol),Pd(PPh₃)₄ (0.000121 mol) and CuI (0.000121 mol) in N,N-diethylethanamine(5 ml) was stirred at 100° C. for 2 hours. Water was added. The mixturewas filtered over celite, washed with EtOAc and extracted aith EtOAc.The organic layer was separated, dried (MgSO₄), filtered and the solventwas evaporated. The residue (0.7 g) was purified by columnchromatography over silica gel (eluent: CH₂Cl₂/CH₃OH 98/2; 15-40 μm).The pure fractions were collected and the solvent was evaporated. Theresidue was crystallized from petroleum ether and diethyl ether. Theprecipitate was filtered off and dried. Yield: 0.1 g of compound (18)(23%); mp. 113° C.

i) Preparation of

A mixture of compound (4) (0.006027 mol) and KF (0.024108 mol) in DMSO(20 ml) was stirred at 140° C. The solvent was evaporated till dryness.The residue was solidified in water and diethyl ether. The mixture wasextracted with diethyl ether. The organic layer was separated, washedwith diethyl ether, washed with a saturated solution of NaCl, dried(MgSO₄), filtered and the solvent was evaporated. The residue (1.7 g)was purified by column chromatography over silica gel (eluent:cyclohexane/EtOAc 85/15; 15-40 μm). Three fractions were collected andtheir solvents were evaporated. The first fraction was crystallized frompetroleum ether. The precipitate was filtered off and dried. Yield: 0.21g of compound (19) (11%); mp. 92° C. The second fraction wascrystallized from petroleum ether. The precipitate was filtered off anddried. Yield: 0.33 g of compound (20) (17%); mp. 114° C.,

j) Preparation of

A mixture of compound (4) (0.003013 mol), acetyl chloride (0.003315 mol)and sodium iodide (0.006027 mol) in CH₃CN (10 ml) was stirred andrefluxed for 1 hour. K₂CO₃ 10% was added. The mixture was extracted withCH₂Cl₂. The organic layer was separated, dried (MgSO₄), filtered and thesolvent was evaporated. The residue (1 g) was purified by columnchromatography over silica gel (eluent: cyclohexane/EtOAc 80/20; 15-40μm). Two fractions were collected and their solvents were evaporated.The first fraction was crystallized from petroleum ether. Theprecipitate was filtered off and dried. Yield: 0.112 g of compound (21);mp. 110° C.,

k) Preparation of

A mixture of compound (21) (0.000898 mol), trimethylsilanecarbonitrile(0.001347 mol) and Pd(PPh₃)₄ (0.00009 mol) in N,N-diethylethanamine (5ml) was stirred at 100° C. for 2 hours. Water was added. The mixture wasextracted with EtOAc. The organic layer was separated, dried (MgSO₄).filtered and the solvent was evaporated. The residue (0.4 g) waspurified by column chromatography over silica gel (eluent:cyclohexane/EtOAc 80/20; 15-40 μm). The pure fractions were collectedand the solvent was evaporated. The residue (0.18 g, 62%) wascrystallized from petroleum ether. The precipitate was filtered off anddried. Yield: 0.13 g of compound (22) (45%); mp. 138° C.

l) Preparation of

A mixture of compound (4) (0.00603 mol), Pd(OAc)₂ (0.000603 mol), PPh₃(0.00904 mol) and K₂CO₃ (0.012054 mol) in CO (gas) and methanol (40 ml)was stirred at 90° C. for 8 hours under a 5 bar pressure of CO. H₂O wasadded. The mixture was extracted with EtOAc. The organic layer wasseparated, dried (MgSO₄), filtered and the solvent was evaporated. Theresidue (6 g) was purified by column chromatography over silica gel(eluent: CH₂Cl₂/CH₃OH 100/0 to 98/2; 15-35 μm). Four fractions (F1-F4)were collected and the solvent was evaporated. Yield: 0.13 g (cis) F1;0.02 g F2 (cis, compound 25); 0.055 g F3 (trans, 3%) and 0.1 g F4(trans; compound 26).

F1 was crystallized from petroleum ether. The precipitate was filteredoff and dried. Yield: 0.03 g of compound (23) (1%); mp. 91° C.

F3 was crystallized from petroleum ether. The precipitate was filteredoff and dried. Yield: 0.035 g of compound (24) (1%); mp. 99° C.

m) Preparation of

A mixture of compound (4) (0.009 mol) and Zn (0.027 mol) in acetic acid(30 ml) was stirred at 60° C. for 4 hours, filtered over celite, washedwith CH₂Cl₂, evaporated till dryness, solubilized in CH₂Cl₂ and washedwith K₂CO₃ 10%. The organic layer was separated, dried (MgSO₄), filteredand the solvent was evaporated. The residue (4 g) was purified by columnchromatography over silica gel (eluent: cyclohexane/EtOAc 75/25; 15-40μm). One fraction was collected and the solvent was evaporated. Thisfraction (1 g 37%) was crystallized from petroleum ether. Theprecipitate was filtered off and dried. Yield: compound (25); mp. 88° C.

n) Preparation of

A mixture of compound (4) (0.001502 mol), Sn(CH₃)₄ (0.003004 mol) andPd(PPh₃)₄ (0.00015 mol) in methylbenzene (5 ml) was stirred and refluxedfor 3 hours. K₂CO₃ 10% was added. The mixture was extracted with EtOAc.The organic layer was separated, dried (MgSO₄), filtered and the solventwas evaporated. The residue (0.7 g) was purified by columnchromatography over silica gel (eluent: cyclohexane/EtOAc 85/15; 15-40μm). Two fractions (F1 and F2) were collected and their solvents wereevaporated. Yield: 0.27 g (F1, starting material) and 0.14 g (F2). F2was crystallized from pentane and petroleum ether. The precipitate wasfiltered off and dried. Yield: 0.08 g of compound (27) (17%); mp. 110°C.

o) Preparation of

A mixture of compound (4) (0.001507 mol), tributylethenylstannane(0.002260 mol) and Pd(PPh₃)₄ (0.000151 mol) in dioxane (5 ml) wasstirred at 80° C. for 8 hours. Water was added. The mixture was filteredover celite, washed with EtOAc and extracted with EtOAc. The organiclayer was separated, dried (MgSO₄), filtered and the solvent wasevaporated. The residue (0.65 g) was purified by column chromatographyover silica gel (eluent: cyclohexane/EtOAc 90/10; 15-40 μm). The purefractions were collected and the solvent was evaporated. The residue wascrystallized from petroleum ether. The precipitate was filtered off anddried. Yield: 0.07 g of compound (28) (14%); mp. 108° C.

p) Preparation of

A mixture of compound (5) (0.001507 mol),triphenyl(phenylmethyl)stannane (0.002260 mol) and Pd(PPh₃)₄ (0.000151mol) in dioxane (5 ml) was stirred at 80° C. for 8 hours. Water wasadded. The mixture was extracted with EtOAc. The organic layer wasseparated, dried (MgSO₄), filtered and the solvent was evaporated. Theresidue (1.4 g) was purified by column chromatography over silica gel(eluent: CH₂Cl₂/EtOAc 96/4; 15-40 μm). The pure fractions were collectedand the solvent was evaporated. The residue (0.38 g) was crystallizedfrom petroleum ether. The precipitate was filtered off and dried. Yield:0.16 g of compound (29) (28%); mp. 112° C.

q) Preparation of

A mixture of compound (4) (0.001507 mol), tributyl-2-thienylstannane(0.00226 mol) and Pd(PPh₃)₄ (0.0001507 mol) in dioxane (5 ml) wasstirred at 80° C. for 8 hours. K₂CO₃ 10% was added. The mixture wasextracted with EtOAc. The organic layer was separated, dried (MgSO₄),filtered and the solvent was evaporated. The residue (1.7 g) waspurified by column chromatography over silica gel (eluent:cyclohexane/EtOAc 85/15; 15-40 μm). The pure fractions were collectedand the solvent was evaporated. The residue (0.65 g) was crystallizedfrom diethyl ether. The precipitate was filtered off and dried. Yield:0.35 g of compound (30) (61%); mp. 142° C.

r) Preparation of

A mixture of compound (4) (0.0015 mol), 3-thienyl boronic acid (0.00226mol), Pd(PPh₃)₄ (0.00015 mol) and dioxane was stirred and refluxed for24 hours. K₂CO₃ 10% was added. The mixture was extracted with EtOAc. Theorganic layer was separated, dried (MgSO₄), filtered and the solvent wasevaporated. The residue (0.8 g) was purified by column chromatographyover silica gel (eluent: cyclohexane/EtOAc 80/20; 15-40 μm). The purefractions were collected and the solvent was evaporated. The residue(0.4 g, 70%) was crystallized from petroleum ether. The precipitate wasfiltered off and dried. Yield: 0.39 g of compound (31) (68%); mp. 113°C.

s) Preparation of

A mixture of compound (4) (0.003 mol), glycine methyl estermonohydrochloride (0.0066 mol) and Pd(PPh)₄ (0.0003 mol) in Et₃N (2 ml)and toluene (10 ml) was stirred at 100° C. under 5 bar pressure of COfor 8 hours, filtered over celite, washed with CH₂Cl₂ and evaporated.The residue (2 g) was purified by column chromatography over silica gel(eluent: cyclohexane/EtOAc 80/20; 75-35 μm). One fraction was collectedand the solvent was evaporated. This fraction (1 g 80%) was crystallizedfrom diethyl ether. The precipitate was filtered off and dried.Yielding: 0.46 g of compound (32) (37%).

t) Preparation of

A mixture of compound (4) (0.003 mol) and hydrazinecarboxaldehyde(0.0045 mol) in 1-butanol (15 ml) was stirred and refluxed overnight,poured out into water and extracted with CH₂Cl₂. The organic layer wasseparated, dried (MgSO₄), filtered and the solvent was evaporated. Theresidue was purified by column chromatography over silica gel (eluent:CH₂Cl₂/CH₃OH/NH₄OH 95/5/0.1; 15-40 μm). Two fractions (F1 and F2) werecollected and their solvents were evaporated. Yield: 0.3 g F1 and 0.3 gF2.

F1 was crystallized from CH₃CN and diethyl ether. The precipitate wasfiltered off and dried. Yield: 0.102 g of compound (33); mp. 224° C.

F2 was crystallized from CH₃CN and diethyl ether. The precipitate wasfiltered off and dried. Yield: 0.2 g of compound (34); mp. 185° C.,

u) Preparation of

A mixture of compound 4 (0.015 mol) and NaN₃ (0.045 mol) in DMF (50 ml)was stirred at 140° C. for 2 hours. K₂CO₃ 10% was added and the mixturewas extracted with EtOAc. The organic layer was separated, dried(MgSO₄), filtered and the solvent was evaporated. The residue (6 g) waspurified by column chromatography over silica gel (eluent:cyclohexane/EtOAc 60/40; 15-40 μm). The first fraction was collected andthe solvent was evaporated. The residue was crystallized from diethylether. The precipitate was filtered off and dried. Yield: 1.26 g ofcompound (35) (24%); mp. 160° C.

v) Preparation of

A mixture of compound (4) (0.009 mol) and thiourea (0.0099 mol) in ethylalcohol (30 ml) was stirred and refluxed for 12 hours and a solution ofKOH (0.0149 mol) in H₂O (5 ml) was added slowly. The mixture was stirredand refluxed for 1 hour, poured out into water and extracted withCH₂Cl₂. The organic layer was separated, dried (MgSO₄), filtered and thesolvent was evaporated. The residue was purified by columnchromatography over silica gel (cyclohexane/EtOAc 70/30; 15-40 cm). Thepure fractions were collected and the solvent was evaporated. Yielding:1.1 g of F1 (37%) and 0.4 g of F2 (13%). F1 was crystallized from2-propanone. The precipitate was filtered off and dried. Yielding:compound (36). F2 was crystallized from 2-propanone. The precipitate wasfiltered off and dried. Yielding: compound (37).

w) Preparation of

CH₃I (0.0034 mol) was added slowly at room temperature to a solution ofcompound (36) (0.0015 mol), compound (37) (0.0015 mol) and K₂CO₃ (0.0034mol) in acetone (15 ml). The mixture was stirred at room temperature for8 hours. Water was added and the mixture was extracted with CH₂Cl₂. Theorganic layer was separated, dried (MgSO₄), filtered and the solvent wasevaporated. The residue (1.2 g) was purified by column chromatographyover silica gel (eluent: cyclohexane/EtOAc 85/15; 15-40 cm). The purefractions were collected and the solvent was evaporated. Yielding: 0.6 gF1 (57%), and 0.18 g F2 (17%). F1 was crystallized from diethyl ether.The precipitate was filtered off and dried. Yielding: 0.28 g compound(38) (27%). F2 was crystallized from diethyl ether. The precipitate wasfiltered off and dried. Yielding: 0.065 g of compound (39) (6%).

x) Preparation of

A mixture of according to example B3.b (0.0014 mol) in HCl 3N (5 ml) andTHF (5 ml) was stirred and refluxed for a weekend, then poured out intoH₂O, basified with K₂CO₃ and extracted with CH₂Cl₂. The organic layerwas separated, dried (MgSO₄), filtered and the solvent was evaporated.Yielding: 0.5 g of F. This fraction F was crystallized from 2-propanone.The precipitate was filtered off and dried. Yielding: 0.35 g of compound(40) (74%).y) Preparation of

A mixture of compound (5) (0.045 mol), acetamide (0.90013 mol) and K₂CO₃(0.225 mol) was stirred and refluxed at 200° C. for 2 hours, cooled atroom temperature, poured out into H₂O/CH₂Cl₂; and extracted with CH₂Cl₂.The organic layer was separated, dried (MgSO₄), filtered and the solventwas evaporated till dryness. The residue (14.4 g) was crystallized fromCH₃OH. The precipitate was filtered off and dried. The filtrate wasevaporated. The residue (11.27 g) was purified by column chromatographyover silica gel (eluent: CH₂Cl₂/CH₃OH/NH₄OH 96/4/0.1; 15-35 μm). Thepure fractions were collected and the solvent was evaporated. Yielding:4.2 g of compound (188) (65%).

z) Preparation of

A mixture of compound (188) (0.00032 mol), benzoic acid (1.5 equiv.,0.00048 mol), 1-ethyl-3-(3′-dimethylaminopropyl)carbodiimide .HCl (1:1)(1.5 equiv., 0.00048 mol), N-hydroxybenzotriazole (1.5 equiv., 0.00048mol) and Et₃N (1 equiv., 0.00032 mol) in CH₂CL₂ (2 ml) was stirred atroom temperature for 15 hours. The solvent was evaporated. The residuewas purified by HPLC and the product fractions were collected and thesolvent was evaporated. Yield: 0.066 g of compound (205) (49.50%).

aa) Preparation of

A mixture of interm. 20 (0.001507 mol) in HCl 3N (10 ml) and THF (10 ml)was stirred at room temperature for 8 hours, basified with K₂CO₃ 10% andextracted with CH₂Cl₂. The organic layer was separated, dried (MgSO₄),filtered and the solvent was evaporated. The residue (1.2 g) waspurified by column chromatography over silica gel (eluent:cyclohexane/EtOAc 85/15; 15-40 μm). The pure fractions were collectedand the solvent was evaporated. The residue (0.4 g) was crystallizedfrom petroleum ether. The precipitate was filtered off and dried. Yield:0.3 g of compound (6) (58%); mp. 108° C.

ab) Preparation of

A mixture of compound 213 (prepared according to B4) (0.00305 mol) andCH₃ONa (30% in CH₃OH) (0.00916 mol) in CH₃OH (25 ml) was stirred andrefluxed for 15 hours then cooled to room temperature, poured out intoH₂O and extracted with EtOAc. The organic layer was separated, dried(MgSO₄), filtered, and the solvent was evaporated till dryness. Theresidue (1.1 g) was purified by column chromatography over silica gel(eluent: cyclohexane/EtOAc; 40/60; 15-40 μm). Two fractions werecollected and the solvent was evaporated. Yielding: 0.3 g F1 and 0.5 gF2 (50%) F2 was crystallized from diethyl ether/petroleum ether. Theprecipitate was filtered off and dried. Yielding: 0.26 g F1 wascrystallized from pentane. The precipitate was filtered off and dried.Yielding: 0.19 g. This fraction was purified by column chromatographyover silica gel (eluent: CH₂Cl₂/CH₃OH; 98/2; 15-40 cm). The purefractions were collected and the solvent was evaporated. Yielding: 0.11g. This fraction was purified by column chromatography over kromasil(eluent: CH₃OH/H₂O; 70/30). The pure fractions were collected and thesolvent was evaporated. Yielding: 0.09 g. (9%) This fraction wascrystallized from diethyl ether. The precipitate was filtered off anddried. Yielding: 0.08 g of compound 419 (8%).

EXAMPLE B5 Preparation of

Iodomethane (0.00456 mol) was added at 5° C. to a mixture of compound(9) (0.0019 mol), compound (8) (0.0019 mol) and tBuOK (0.00456 mol) inTHF (30 ml) under N₂ flow. The mixture was stirred at room temperatureovernight, poured out into H₂O and extracted with CH₂Cl₂. The organiclayer was separated, dried (MgSO₄), filtered and the solvent wasevaporated. The residue was purified by column chromatography oversilica gel (eluent: cyclohexane/EtOAc 65/35; 15-40 μm). Two fractionswere collected and the solvent was evaporated. Yield: 0.35 g of compound(42) (30%; mp. 125° C.) and 0.35 g of compound (43) (30%; mp. 116° C.).

EXAMPLE B6 a) Preparation of

NaH 60% (0.01068 mol) was added at 0° C. under N₂ flow to a mixture ofcompound (8) and compound (9) (0.0089 mol). The mixture was stirred for30 minutes. Ethyl bromoacetate (0.01068 mol) was added at 0° C. Themixture was stirred at room temperature for 1 hour, hydrolized withwater and extracted with EtOAc. The organic layer was separated, dried(MgSO₄), filtered and the solvent was evaporated. The residue waspurified by column chromatography over silica gel (eluent:cyclohexane/EtOAc 60/40; 15-40 μm). The desired fractions (F1-F4) werecollected and the solvent was evaporated. Yield: 0.11 g F; 0.13 g F2;0.75 g F3 and 0.8 g F4.

F3 was crystallized from diethyl ether. The precipitate was filtered offand dried. Yield: compound (44); mp. 152° C.

F4 was crystallized from diethyl ether. The precipitate was filtered offand dried. Yield: compound (45); mp. 147° C.

b) Preparation of

Bromomethylbenzene (0.007 mol) was added dropwise at 0° C. under N₂ flowto a solution of compound (8) and compound (9) (0.0064 mol) and NaH 60%(0.007 mol) in DMF (40 ml). The mixture was stirred at room temperaturefor 1 hour, hydrolized with water and extracted with EtOAc. The organiclayer was separated, washed with water, dried (MgSO₄), filtered and thesolvent was evaporated. The residue was purified by columnchromatography over silica gel (eluent: cyclohexane/EtOAc 70/30; 15-40μm). The desired fractions (F1-F4) were collected and the solvent wasevaporated. Yield: 0.15 g F1, 0.11 g F2, 0.6 g F3 (23%) and 0.8 g F4.

F3 was crystallized from diethyl ether. The precipitate was filtered offand dried. Yield: 0.13 g of compound (46); mp. 137° C.

F4 was crystallized from DIPE and petroleum ether. The precipitate wasfiltered off and dried. Yield: compound (47); mp. 130° C.

EXAMPLE B7

a) 3-Chlorobenzenecarboperoxoic acid (0.088 mol) was added at 0° C. to asolution of compound (48) (prepared according to example B2) (0.044 mol)in CH₂Cl₂ (200 ml) and the mixture was stirred at room temperature for12 hours. The mixture was washed with K₂CO₃ 10%. The organic layer wasdried (MgSO₄), filtered off and evaporated. The residue wasrecrystallized from (C₂H₅)₂O. Yield: 8.2 g ofcyclohexyl(3-methyl-6-quinolinyl)methanone, 11-oxide (compound 49)(69%).

b) 4-Methyl benzenesulfonyl chloride (0.043 mol) was added to a solutionof compound (49) (0.028 mol) in K₂CO₃ (400 ml) and CH₂Cl₂ (400 ml) andthe mixture was stirred at room temperature for 1 hour. The mixture wasextracted with CH₂Cl₂. The organic layer was dried (MgSO₄), filtered offand evaporated. The residue was recrystallized from (C₂H₅)₂O. Yield:6.64 g of 6-(cyclohexylcarbonyl)-3-methyl-2(1H)-quinolinone (compound50) (85%); mp. 256.1° C.

EXAMPLE B8 a) Preparation of

A mixture of compound (7) (0.0229 mol), hydroxylamine (0.0252 mol) andN,N-diethylethanamine (0.0252 mol) in ethanol (100 ml) was stirred andrefluxed for 6 hours, poured out into water and extracted with CH₂Cl₂.The organic layer was separated, dried (MgSO₄), filtered and the solventwas evaporated. The residue was crystallized from CH₃CN. The precipitatewas filtered off and dried. The residue was purified by columnchromatography over silica gel (eluent: CH₂Cl₂/EtOAc 80/20; 15-40 μm).Two fractions were collected and the solvent was evaporated. Yielding:2.8 g of compound (51) (36%; mp. 133° C.) and 3 g of compound (52) (38%;mp. 142° C.).

c) Preparation of

Hydrazine (0.41 mol) was added at room temperature to a solution ofcompound (7) (0.015 mol) in ethanol (75 ml). The mixture was stirred andrefluxed for 1 night, poured out into water and extracted with CH₂Cl₂.The organic layer was separated, dried (MgSO₄), filtered and the solventwas evaporated. The residue was purified by column chromatography oversilica gel (eluent: CH₂Cl₂/CH₃OH/NH₄OH 98/2/0.1). The pure fractionswere collected and the solvent was evaporated. The residue wascrystallized from diethyl ether. The precipitate was filtered off anddried. Yielding: 0.8 g of compound (53) (15%); mp. 110° C.

EXAMPLE B9 Preparation of

Procedure for compounds 400, 401, 402, 403, 404 and 405. A mixture ofinterm. 21 (prepared according to A11) (0.000269 mol), amantadinehydrochloride (0.000404 mol; 1.5 eq.),N′-(ethylcarbonimidoyl)-N,N-dimethyl-1,3-propanediamine hydrochloride(0.000404 mol; 1.5 equiv.), 1-hydroxy-1H-benzotriazole (0.000404 mol;1.5 equiv.) and Et₃N (0.000269 mol) in CH₂Cl₃ (2 ml) was stirred at roomtemperature for 12 hours. The solvent was evaporated. The residue waspurified by HPLC. The product fractions were collected and the solventwas evaporated. Yield: 0.063 g of compound 520 (46.37%).

EXAMPLE B10 Preparation of

A mixture of intermediate 27 (0.0026 mol) and intermediate 26 (0.0026mol) in EtOH (380 ml) and H₂SO₄ conc. (19 ml) was stirred and refluxedfor 15 hours, the cooled to room temperature, poured out into ice water,basified with K₂CO₃ and extracted with EtOAc. The organic layer wasseparated, dried (MgSO₄), filtered, and the solvent was evaporated. Theresidue (17.9 g) was purified by column chromatography over silica gel(eluent: cyclohexane/EtOAc; 80/20; 15-35 μm). The pure fractions werecollected and the solvent was evaporated. Yielding: 0.85 g of F1, 1.1 gF2 and 11.5 g of F3. F1 and F2 were crystallized separately frompetroleum ether. The precipitate was filtered off and dried. Yielding:0.34 g of compound 233.

EXAMPLE B11 Preparation of

A mixture of compound 22 (prepared according to B4) (0.004 mol) in HCl(3N) (20 ml) and THF (20 ml) was stirred and refluxed for 8 hours,poured out on ice, basified with NH₄OH and extracted with CH₂Cl₂. Theorganic layer was separated, dried (MgSO₄), filtered, and the solventwas evaporated. The residue (1.2 g) was purified by columnchromatography over silica gel (eluent: CH₂Cl₂/CH₃OH/NH₄OH; 93/7/0.5;15-40 μm). Two fractions were collected and the solvent was evaporated.Yielding: 0.5 g F1 (41%) and 0.4 g of F2. F1 was crystallized frompetroleum ether. The precipitate was filtered off and dried. Yielding:0.17 g of compound 511 (14%).

EXAMPLE B12 Preparation of

A mixture of compound 524 (prepared according to B9a) (0.0018 mol) andKOH 85% (0.0094 mol) in EtOH (15 ml) was stirred and refluxed for 24hours, poured out into H₂O and extracted with CH₂Cl₂. The organic layerwas separated, dried (MgSO4), filtered, and the solvent was evaporated.The residue was purified by column chromatography over silica gel(eluent: CH₂Cl₂/Cyclohexane 80/20; 15-40 μm). Two fractions werecollected and the solvent was evaporated. Yielding: 0.35 g F1 (64%) and0.17 g (SM) F1 was crystallized from diethyl ether. The precipitate wasfiltered off and dried. Yielding: 0.33 g of compound 514 (60%) (mp.:185° C.).

EXAMPLE B13 Preparation of

A mixture of interm. 28 (0.019 mol), 2-benzofuranylboronic acid (0.028mol), Pd(PPh₃)₄ (0.001 mol) and BHT (a few quantity) in dioxane (25 ml)and Na₂CO₃ [2] (25 ml) was stirred and refluxed for 8 hours andextracted with EtOAc. The aqueous layer was basified with NH₄OH andextracted with CH₂Cl₂. The organic layer was separated, dried (MgSO₄),filtered, and the solvent was evaporated. The residue (3.6 g) waspurified by column chromatography over silica gel (eluent: CH₂Cl₂/CH₃OH99/1; 15-40 μm). The pure fractions were collected and the solvent wasevaporated. Yielding: 1.8 g (33%). This fraction was crystallized from2-propanone/diethyl ether. The precipitate was filtered off and dried.Yielding: 0.39 g of compound 515 (7%) (mp.: 134° C.).

EXAMPLE B14 Preparation of

Triethylsilane (0.0012 mol) was added slowly at room temperature to asolution of interm. 32 (0.004 mol) in CF₃COOH (5 ml) and AcOH (10 ml).NaBH₄ (0.0012 mol) was added portionwise under N₂ flow. The mixture wasstirred at room temperature for 8 hours, poured out on ice, basifiedwith K₂CO₃ and extracted with CH₂Cl₂. The organic layer was separated,dried (MgSO₄), filtered, and the solvent was evaporated. The residue(1.2 g) was purified by column chromatography over silica gel (eluent:CH₂Cl₂/CH₃OH 99/1; 15-40 μm). Two fractions were collected and thesolvent was evaporated. Yielding: 0.5 g F1 (43%) and 0.4 g F2. F1 wasdissolved in iPrOH. HCl/iPrOH (1 eq) were added. The precipitate wasfiltered off and dried; Yielding: 0.32 g of compound 526 (mp.: 248° C.).

EXAMPLE B15 Preparation of

A mixture of interm. 33 (0.082 mol) and 3-chloro-2-ethyl-2-butenal(0.098 mol) in AcOH (200 ml) was stirred and refluxed for 8 hours. Thesolvent was evaporated till dryness. The residue was dissolved in CH₂Cl₂and washed with K₂CO₃ 10%. The organic layer was separated, dried(MgSO₄), filtered, and the solvent was evaporated. The residue (27 g)was purified by column chromatography over silica gel (eluent:CH₂Cl₂/EtOAc 95/5 to 92/8; 15-35 μm). Two fractions were collected andthe solvent was evaporated. Yielding: 0.7 g of F1 and 5.3 g F2. F1 wascrystallized from 2-propanone/diethyl ether. The precipitate wasfiltered off and dried. Yielding: 0.25 g of compound 471 (2%) (mp.: 140°C.).

EXAMPLE B16 Preparation of

nBuLi (0.0417 mol) was added dropwise at −78° C. to a solution ofinterm. 35 (prepared according to A17.b) (0.0379 mol) in THF (200 ml)under N₂ flow. The mixture was stirred for 30 minutes. A solution of4-bromo-N-methoxy-N-methylbenzeneacetamide (0.0568 mol) in THF (100 ml)was added dropwise at −78° C. The mixture was stirred from −78° C. to 0°C., poured out into H₂O and extracted with EtOAc. The organic layer wasseparated, dried (MgSO₄), filtered, and the solvent was evaporated tilldryness. The residue (20.9 g) was purified by column chromatography oversilica gel (eluent: toluene/EtOAc 60/40 to 50/50; 15-35 μm). Twofractions were collected and the solvent was evaporated. Yielding: 4 gof fraction 1 and 4 g of fraction 2 (28%). Fraction 2 was crystallizedfrom diethyl ether. The precipitate was filtered off and dried.Yielding: 1 g compound 528 (m.p. 195° C.).

Tables 1 to 8 list the compounds of formula (I-A) and (I-B) which wereprepared according to one of the above examples. TABLE 1

Co. Ex. physical no. no. R² R³ R⁴ R¹ data 54 B2 Cl ethyl H

— 3 B3a Cl ethyl H

mp. 145° C. 55 B3b Cl ethyl H

mp. 131° C. 56 B3b Cl ethyl H

mp. 104° C. 57 B3b Cl ethyl H phenylethyl mp. 100° C. 58 B3b Cl ethyl H

mp. 126° C. 59 B3b Cl ethyl H

mp. 150° C. 60 B3b Cl ethyl H

mp. 138° C. 61 B3b OCH₃ ethyl H

— 62 B3b OCH₃ ethyl H

mp. 130° C. 63 B3b OCH₃ ethyl H

mp. 116° C. 64 B3b Cl ethyl H —(CH₂)₂—O—CH₃ mp. 82° C. 65 B3b OCH₃ ethylH 1-methylcyclohexyl mp. 82° C. 66 B3b OCH₃ ethyl H 3-methoxycyclohexyltrans; mp. 94° C. 67 B3b OCH₃ ethyl H 3-methoxycyclohexyl cis; mp. 108°C. 68 B3b OCH₃ ethyl H 4-(methylethoxy)- (A), mp. cyclohexyl 82° C. 69B3b OCH₃ ethyl H 4-[C(CH₃)₃]cyclohexyl cis; mp. 92° C. 70 B3b OCH₃ ethylH 4-[C(CH₃)₃]cyclohexyl trans; mp. 108° C. 71 B3b OCH₃ ethyl H4-methylcyclohexyl (B), mp. 92° C. 72 B3b OCH₃ ethyl H4-methylcyclohexyl (A), mp. 80° C. 2 B2 Cl ethyl H CH₂—CH(CH₃)₂ mp. 82°C. 73 B3b Cl ethyl H —CH₂—O—C₂H₅ mp. 82° C. 48 B2 H methyl H cyclohexyl— 74 B4 I ethyl H

— 75 B4 I ethyl H

mp. 124° C. 76 B4 I ethyl H

mp. 138° C. 77 B4 I ethyl H

mp. 120° C. 78 B4 CN ethyl H

mp. 128° C. 79 B4 CN ethyl H

mp. 136° C. 80 B4 CN ethyl H

mp. 120° C. 81 B4 CN ethyl H

mp. 139° C. 82 B4 methyl ethyl H

mp. 106° C. 83 B4 methyl ethyl H

mp. 149° C. 84 B4 methyl ethyl H

mp. 118° C. 85 B4 methyl ethyl H

mp. 180° C. 86 B4 methyl ethyl H phenylethyl mp. 53° C. 87 B4 methylethyl H

mp. 87° C. 88 B4 methyl ethyl H —CH₂—CH(CH₃)₂ mp. 68° C. 89 B4 methylethyl H

mp. 120° C. 31 B4 3-thiazolyl ethyl H 4-methoxycyclohexyl cis; 113° C.90 B3b OCH₃ H H 4-methoxycyclohexyl trans, mp. 126° C. 91 B3b OCH₃ H H4-methoxycyclohexyl cis, mp. 100° C. 92 B3b OCH₃ H CH₃4-methoxycyclohexyl cis; mp. 120° C. 93 B3b OCH₃ H CH₃4-methoxycyclohexyl trans; mp. 111° C. 94 B3b OCH₃ methyl H4-methoxycyclohexyl cis, mp. 96° C. 95 B3b OCH₃ phenyl H4-methoxycyclohexyl cis; HCl (1:1), mp. 138° C. 96 B3b OCH₃ propyl H4-methoxycyclohexyl trans; mp. 118° C. 97 B3b OCH₃ propyl H4-methoxycyclohexyl cis; mp. 108° C. 98 B3b OCH₃ methyl H4-methoxycyclohexyl cis; mp. 104° C. 99 B4 N(CH₃)₂ ethyl H

(B); mp. 102° C. 100 B3b Cl ethyl H

mp. 114° C. 101 B4 methyl ethyl H 4-butoxycyclohexyl cis; mp. 86° C. 102B3b Cl ethyl H

mp. 78° C. 103 B3b Cl ethyl H

mp. 91° C. 104 B4 N(CH₃)₂ ethyl H

mp. 103° C. 105 B4 N(CH₃)₂ ethyl H

mp. 170° C. 106 B3b Cl ethyl H

mp. 137° C. 107 B3b Cl ethyl H

mp. 137° C. 108 B4 methyl ethyl ethyl 4-methoxycyclohexyl cis; mp. 91°C. 109 B4 methyl ethyl H 4-ethoxycyclohexyl trans; mp. 110 B4 methylethyl H

mp. 90° C. 111 B4 methyl ethyl H

mp. 94° C. 112 B4 methyl ethyl H

mp. 176° C. 113 B4 methyl ethyl H

mp. 106° C. 114 B4 propyl H H 4-methoxycyclohexyl cis; mp. 74° C. 115 B4methyl ethyl H 4-ethoxycyclohexyl cis; mp. 108° C. 116 B4 methyl ethyl H

mp. 110° C. 117 B3b Cl ethyl H

mp. 124° C. 118 B3b Cl ethyl H

mp. 107° C. 119 B3b Cl ethyl H

mp. 129° C. 120 B4 methyl ethyl H

mp. 106° C. 41 B3b Cl ethyl H

trans; mp. 157° C. 182 B3b methyl ethyl H

cis; mp. 170° C. 183 B3b methyl ethyl H

trans; mp. 144° C. 184 B3b methyl ethyl H

mp. 138° C. 185 B3b Cl ethyl H

mp. 120° C. 186 B3b Cl ethyl H

187 B3b methyl ethyl H

mp. 162° C. 216 B4 CC≡N ethyl H

mp.: 160° C. 217 B4 methyl ethyl H

.ethanedioate (1:1); mp.: 143° C. 218 B4 I ethyl H

mp.: 102° C. 219 B4 CC═N ethyl H

mp.: 115° C. 220 B4 Cl ethyl H

(A); mp.: 107° C. 221 B4 Cl ethyl H

(B); mp.: 113° C. 222 B4 I ethyl H

mp.: 206° C. 223 B4 Cl ethyl H

(trans); mp.: 117° C. 224 B4 methyl ethyl H

(A); mp.: 103° C. 225 B2 Cl ethyl H

mp.: 94° C. 226 B3b Cl ethyl H

(trans); mp.: 157° C. 227 B3c methoxy

H

mp.: 204° C. 228 B4 Cl ethyl H

(A); mp.: 136° C. 229 B3b n-propyl H H

(trans);.HCl (1:1); mp.; 150° C. 230 B3b Cl ethyl H

mp.: 116° C. 231 B3b Cl ethyl H

mp.: 120° C. 232 B3b Cl ethyl H

mp.: 112° C. 233 B10 i-propyl H C(═O)O—C₂H₅

(cis); mp.: 91° C. 234 B4 methyl ethyl H

mp.: 122° C. 235 B4 methyl ethyl H

mp.: 106° C. 236 B4 methyl ethyl H

mp.: 104° C. 237 B4 methyl ethyl H

mp.: 90° C. 238 B4 methyl H H

(cis); mp.: 80° C. 239 B3b Cl ethyl H

(trans); mp.: 126° C. 240 B3b Cl ethyl H

(cis); mp.: 128° C. 241 B4 methyl ethyl H

(A); mp.: 90° C. 242 B4 methyl ethyl H

(B); mp.: 110° C. 243 B3b Cl ethyl H

mp.: 134° C. 244 B3b Cl ethyl H

mp.: 127° C. 245 B4 NHC(═O)NH₂ ethyl H

(cis); mp.: 176° C. 246 B4 methyl ethyl H

(B) 247 B3b Cl ethyl H

mp.: 92° C. 248 B4 methyl ethyl H

(A); mp.: 80° C. 249 B3b Cl ethyl H

(B); mp.: 138° C. 250 B4 methyl ethyl H

(trans); mp.: 118° C. 251 B4 methyl ethyl H

(B);.HCl(1:1) 252 B3b Cl ethyl H

(A) 253 B3b Cl ethyl H

(B) 254 B3b methyl ethyl H

mp.: 74° C. 255 B4 methyl ethyl H

(cis); mp.: 68° C. 256 B4 methyl ethyl H

mp.: 210° C. 257 B4 methyl ethyl H

mp.: 113° C. 258 B4 methyl ethyl H

mp.: 92° C. 259 B3b methyl ethyl H

mp.: 115° C. 260 B3b methyl ethyl H

mp.: 60° C. 261 B3b Cl ethyl H

(A); mp.: 86° C. 262 B3b Cl ethyl H

(B); mp.: 101° C. 263 B3b methyl ethyl H

mp.: 130° C. 264 B3b Cl ethyl H

(A); mp.: 124° C. 265 B3b Cl ethyl H

(B); mp.: 126° C. 266 B4 N(CH₃)₂ ethyl H

(trans); mp.: 102° C. 267 B4 N(CH₃)₂ ethyl H

(cis);.HCl(1:1); mp.: 170° C. 268 B4 methyl ethyl H

(A);.HCl(1:1); mp.: 206° C. 269 B4 methyl ethyl H

mp.: 104° C. 270 B3b methyl ethyl H

mp.: 117° C. 271 B4 NHC₂H₅OCH₃ ethyl H

— 272 B4 methyl ethyl H

— 273 B4 NH₂ ethyl H

— 274 B3b Cl ethyl H

— 275 B3b Cl ethyl H

mp.: 99° C. 276 B3b Cl ethyl H

mp.: 95° C. 277 B4 methyl ethyl H

mp.: 105° C. 278 B3b Cl ethyl H

mp.: 141° C. 279 B4 Cl ethyl H

mp.: 168° C. 280 B4 Cl ethyl H

— 281 B4 Cl ethyl H

mp.: 140° C. 282 B4 Cl ethyl H

mp.: 169° C. 283 B4 methyl ethyl H

mp.: 96° C. 284 B3b Cl CH₂N(CH₃)₂ H

mp.: 115° C. 285 B4 methyl ethyl H

mp.: 133° C. 286 B4 methyl CH₂OCH₃ H

(trans); mp.: 106° C. 287 B4 methyl CH₂N(CH₃)₂ H

(cis); mp.: 110° C. 288 B3b Cl n-propyl H

mp.: 110° C. 289 B4 NH₂ ethyl H

mp.: 218° C. 290 B4 methyl n-propyl H

mp.: 90° C. 291 B3b Cl n-propyl H

(cis); mp.: 128° C. 292 B3b Cl n-propyl H

(trans); mp.: 104° C. 293 B3b Cl ethyl H

mp.: 106° C. 294 B4 methyl n-propyl H

(cis); mp.: 94° C. 295 B4 methyl CH₂N(CH₃)₂ H

mp. 83° C. 296 B3b Cl ethyl H

mp.: 99° C. 297 B3b Cl ethyl H

mp.: 110° C. 298 B4 methyl ethyl H

mp.: 93° C. 299 B4 methyl ethyl H

mp.: 105° C. 300 B4 methyl ethyl H

mp.: 114° C. 301 B3b methyl ethyl H

mp.: 143° C. 302 B4 methoxy ethyl H

mp.: 93° C. 303 B4 methyl ethyl H

mp.: 82° C. 304 B4 n-butyl ethyl H

— 305 B3b Cl n-propyl H

mp.: 125° C. 306 B1 methyl C(═O)OC₂H₅ H

mp.: 136° C. 307 B4 methyl n-propyl H

mp.: 81° C. 308 B4 methoxy n-propyl H

mp.: 80° C. 309 B4 I n-propyl H

mp.: 120° C. 310 B3d methyl ethyl H

.HCl(1:1); mp.: 129° C. 311 B3b Cl H H

mp.: 160° C. 312 B3b Cl H H

(trans); mp.: 145° C. 313 B3b Cl H H

mp.: 103° C. 314 B4 n-propyl n-propyl H

.HCl(1:1); mp.: 150° C. 315 B4 n-propyl ethyl H

.HCl(1:1) 316 B4 n-propyl H H

.HCl(1:1); mp.: 140° C. 317 B3b Cl H H

mp.: 168° C. 318 B4 methyl n-propyl H

.HCl(1:1); mp.: 200° C. 509 B3b Cl ethyl H

— 510 B4 methyl ethyl H

.H₂O(1:1) 513 B4 methyl ethyl H

— 516 B4 Cl ethyl H

mp.: 120° C. 517 B4 I ethyl H CH₂CH(CH₃)₂ — 518 B4 Cl ethyl H

— 519 B4 Cl ethyl H

(A + B) 521 B4 I ethyl H

— 522 B4 methyl ethyl H

(A) 1 B4 methyl ethyl H

(A) 525 B4 Cl ethyl H

527 B4 F ethyl H

mp.: 116° C.

TABLE 2

Co. Ex. no. no. R² X physical data 5 B3b Cl O trans; mp. 120° C. 121 B3b1-piperidinyl O cis; HCl (1:1) 122 B3b 1-piperidinyl O trans; HCl (1:1);mp. 128° C. 123 B3b 4-thiomorpholinyl O cis; mp. 105° C. 124 B3b4-thiomorpholinyl O trans; mp. 115° C. 125 B3b 4-morpholinyl O trans;mp. 118° C. 126 B3b 4-morpholinyl O cis; mp. 118° C. 127 B3b —N(CH₃)₂ Otrans; mp. 96° C. 128 B3b —N(CH₃)₂ O cis; mp. 114° C. 4 B3b Cl O cis;mp. 123° C. 8 B3c OCH₃ O trans, mp. 68° C. 7 B3c OCH₃ O cis, mp. 116° C.6 B4 acetyl O trans; mp. 108° C. 129 B4 acetyl O cis; mp. 106° C. 11 B4NH—(CH₂)₂—OCH₃ O trans; mp. 107° C. 10 B4 NH—(CH₂)₂—OCH₃ O cis; mp. 115°C. 12 B4 NH—(CH₂)₂—SCH₃ O cis; mp. 120° C. 13 B4 NH—(CH₂)₂—SCH₃ O trans;mp. 125° C. 14 B4 —C≡C—Si(CH₃)₃ O cis; mp. 114° C. 16 B4 —C≡C—Si(CH₃)₃ Otrans; mp. 108° C. 15 B4 —C≡CH O cis; mp. 132-133° C. 17 B4 —C≡CH Otrans; mp. 128° C. 18 B4 —C≡C—CH₂OH O cis; mp. 113° C. 130 B4 —C≡C—CH₂OHO trans; mp. 108° C. 19 B4 F O cis; mp. 92-99° C. 20 B4 F O trans; mp.114° C. 21 B4 I O cis; mp. 110° C. 22 B4 CN O cis; mp. 137-138° C. 26 B4H O trans 23 B4 —C(═O)—OCH₃ O cis; mp. 91° C. 24 B4 —C(═O)—OCH₃ O trans;mp. 99° C. 25 B4 H O cis; mp. 88° C. 27 B4 methyl O cis; mp. 110-112° C.131 B4 methyl O trans; mp. 25° C. 28 B4 ethenyl O cis; mp. 108° C. 132B4 ethenyl O trans; mp. 103° C. 29 B4 phenyl O trans; mp. 112° C. 30 B42-thienyl O cis; 142° C. 133 B4 2-thiazolyl O cis; 108° C. 134 B42-furanyl O cis; mp. 105° C. 51 B8a OCH₃ N—OH [1α(A),4α]; mp. 133° C. 52B8a OCH₃ N—OH [1α(B),4α]; mp. 142° C. 53 B8b OCH₃ NNH₂ [1α(Z),4α]; mp.110° C. 135 B4 NH₂ O cis; mp. 203° C. 136 B4 NH₂ O trans; mp. 202° C.137 B4 —C(═O)—OCH(CH₃)₂ O cis; mp. 105° C. 138 B4 —C(═O)—OCH(CH₃)₂ Otrans; mp. 88° C. 38 B4 SCH₃ O cis; mp. 124° C. 39 B4 SCH₃ O trans; mp.116° C. 32 B4

O cis; mp. 130° C. 139 B4 ethyl O cis; mp. 180° C. 188 B4 NH₂ O cis +trans 189 B4

O cis; mp. 154° C. 190 B4

O trans; mp. 156° C. 191 B4

O cis; mp. >260° C. 192 B4

O .H2O(1:1); trans; mp. 248° C. 193 B4

O cis; mp. 224° C. 194 B4

O trans; mp. 234° C. 195 B4

O cis; mp. 108° C. 196 B4

O trans; mp. 127° C. 197 B4

O cis; mp. 150° C. 198 B4

O trans; mp. 90° C. 199 B4

O LC/MS [M + H]⁺; 475.4 200 B4

O LC/MS [M + H]⁺; 464.3 201 B4

O LC/MS [M + H]⁺; 523.3 202 B4

O LC/MS [M + H]⁺; 465.3 203 B4

O LC/MS [M + H]⁺; 475.4 204 B4

O LC/MS [M + H]⁺; 465.3 205 B4

O 319 B4

O (cis);.ethanedioate(1:1); mp.: 160° C. 320 B4

O (cis); mp.: 150° C. 321 B4 methoxy CH₂ (cis);.HCl(1:1); mp.: 118° C.322 B4 n-butyl O (cis);.HCl(1:1); mp.: 158° C. 323 B4

O — 324 B4

O — 325 B4

O — 326 B4

O — 327 B4

O — 328 B4

O — 329 B4

O — 330 B4

O — 331 B4

O — 332 B4

O — 333 B4

O — 334 B4

O — 335 B4

O — 336 B4

O — 337 B4

O — 338 B4

O — 339 B4

O — 340 B4

O — 341 B4

O — 342 B4

O — 343 B4

O — 344 B4

O — 345 B4

O — 346 B4

O — 347 B4

O — 348 B4 CH₂OPC(═O)CH₃ O (cis); mp.: 74° C. 349 B4

O — 350 B4

O — 351 B4

O — 352 B4

O — 353 B4

O (A);.HCl(1:2).H2O(1:1); mp.: 166° C. 354 B4

O (cis) 355 B4

O — 356 B4

O — 357 B4

O — 358 B4

O — 359 B4

O — 360 B4

O — 361 B4

O — 362 B4

O — 363 B4

O — 364 B4

O — 365 B4

O — 366 B4

O — 367 B4

O — 368 B4

O — 369 B4

O — 370 B4

O — 371 B4

O — 372 B4

O — 373 B4

O — 374 B4

O — 375 B4

O — 376 B4

O — 377 B4

O — 378 B4

O — 379 B4

O — 380 B4

O — 381 B4

O — 382 B4

O — 383 B4

O (cis); mp.: 148° C. 384 B4

O (trans); mp.: 141° C. 385 B4

O mp.: 130° C. 386 B4

O (cis); mp.: 140° C. 387 B4

O (trans); mp.; 155° C.

TABLE 3

Co. Ex. no. no. Y. R¹ physical data 140 B4 O

mp. 220° C. 141 B4 O

mp. 213° C. 142 B4 O

mp. 148° C. 143 B4 O 1-methylcyclohexyl mp. 195-210° C. 144 B4 O3-methoxycyclohexyl cis; mp. 156° C. 145 B4 O 3-methoxycyclohexyl trans;mp. 156-163° C. 146 B4 O 4-(dimethylethyl)cyclohexyl mp. 230° C. 147 B4O 4-(methylethoxy)cyclohexyl mp. 186° C. 148 B4 O 4-methylcyclohexyltrans; mp. 214° C. 36 B4 S 4-methoxycyclohexyl cis; mp. 224° C. 37 B4 S4-methoxycyclohexyl trans; mp. 220° C. 149 B4 O

mp. 188° C. 40 B4 O

mp. 192° C. 150 B4 O

cis; mp. 226° C. 151 B4 O

trans; mp. 226° C. 152 B4 O

mp. 213° C. 153 B4 O

mp. 200° C. 154 B4 O

mp. 210° C. 155 B4 O 4,4-dimethylcyclohexyl mp. 242° C. 388 B4 OCH₂CH(CH₃)₂ mp. 189° C. 389 B4 O

mp. 228° C. 390 B4 O

mp. 197° C. 391 B4 O

mp. 145° C. 392 B4 O

mp. 192° C. 393 B4 O

(B); mp.: 224° C. 394 B4 O

(A); mp.: 201° C. 395 B4 O

(A); mp.: 207° C. 396 B4 O

mp.: 212° C. 397 B4 O

(B); mp.: 238° C. 398 B4 O

mp.: 234° C. 399 B4 O

(cis); mp.: 192° C.

TABLE 4

Co. Ex. no. no. R³ R⁴ R⁵ R physical data 156 B4 ethyl H H OCH₃ trans;mp. 252° C. 157 B4 H H H OCH₃ (cis + trans); mp. 244° C. 158 B4 H methylH OCH₃ cis; mp. >260° C. 159 B4 methyl H H OCH₃ cis; mp. 254° C. 160 B4methyl H H OCH₃ trans; mp. >260° C. 161 B4 propyl H H OCH₃ mp. 208° C.162 B4 propyl H H OCH₃ trans; mp. 232° C. 9 B4 ethyl H H OCH₃ cis; mp.224-226° C. 43 5 ethyl H CH₃ OCH₃ trans; mp. 116° C. 42 5 ethyl H CH₃OCH₃ cis; mp. 125° C. 44 6 ethyl H CH₂—COOC₂H₅ OCH₃ 152° C. 45 B4 ethylH CH₂—COOC₂H₅ OCH₃ trans; mp. 147° C. 46 B4 ethyl H benzyl OCH₃ cis; mp.137° C. 47 B4 ethyl H benzyl OCH₃ trans; mp. 130° C. 50 7 methyl H H Hmp. 256.1° C. 163 B4 ethyl ethyl H OCH₃ cis; mp. 221° C. 164 B4 ethylethyl H OCH₃ cis; mp. 221° C. 165 B4 ethyl ethyl H OCH₃ trans; mp. 215°C. 166 B4 ethyl H

OCH₃ LC/MS[M + H]⁺; 429.4 167 B4 ethyl H

OCH₃ LC/MS[M + H]⁺; 451.3 168 B4 H H H OCH₃ cis; mp. 106° C. 169 B4ethyl H

OCH₃ LC/MS[M + H]⁺; 409.3 400 B9 ethyl H

OCH₃ — 401 B9 ethyl H

OCH₃ — 402 B9 ethyl H

OCH₃ — 403 B9 ethyl H

OCH₃ — 404 B9 ethyl H

OCH₃ — 405 B9 ethyl H

OCH₃ — 406 B4 ethyl H

OCH₃ — 407 B4 ethyl H

OCH₃ — 408 B4 ethyl H

OCH₃ — 409 B3b

H H OCH₃ mp.: 168° C. 410 B4 CH₂OCH₃ H H OCH₃ mp.: 194° C. 508 B4 ethylH

OCH₃ — 520 B9 ethyl H

OCH₃ —

TABLE 5

Co. Ex. no. no. R⁴ R¹ X physical data 33 B4 H methoxycyclohexyl CH cis;mp. 224° C. 34 B4 H methoxycyclohexyl CH trans; mp. 185° C. 35 B4 Hmethoxycyclohexyl N cis; mp. 160-172° C. 170 B4 H methoxycyclohexyl Ntrans; mp. 146° C. 171 B4 H

N (B); mp. 165° C. 172 B4 H methylcyclohexyl N cis + trans; mp. 143° C.173 B4 ethyl methoxycyclohexyl N cis; mp.: 126° C. 411 B4 H

N mp.: 109° C. 412 B4 H

N mp.: 180° C. 413 B4 H

N (A) 414 B4 H

N mp.: 156° C.

TABLE 6

Co. Ex. no. no. R L physical data 49 B7 H

— 174 B3b OCH₃

cis; mp. 115° C. 175 B3b OCH₃

trans; mp. 141° C. 176 B3b OCH₃

cis; mp. 149° C. 177 B3b OCH₃

mp. 126° C. 178 B3b OCH₃

trans; mp. 160° C. 179 B3b OCH₃

cis; mp. 119° C. 180 B3b OCH₃

trans; mp. 124° C. 181 B3b OCH₃

trans; mp. 92° C. 206 B3b OCH₃

cis; m.p. 144° C. 207 B3b OCH₃

trans; m.p. 125° C. 208 B3b OCH₃

cis; m.p. 127° C. 209 B3b OCH₃

cis; m.p. 101° C. 210 B3b OCH₃

cis; m.p. 104° C. 211 B3b OCH₃

trans; m.p. 134° C. 212 B4 OCH₃

cis; m.p. 141° C. 213 B4 OCH₃

trans; m.p. 215° C. 214 B4 OCH₃

cis; m.p. 139° C. 215 B3b OCH₃

trans 415 B3b OCH₃

(cis); mp.: 136° C. 416 B3b OCH₃

(cis) 417 B4 OCH₃

(cis); mp.: 149° C. 418 B3b OCH₃

(trans); mp.: 132° C. 419 B4 OCH₃

(cis); mp.: 217° C. 420 B3b OCH₃

(cis); HCl(1:1); mp.: 200° C. 421 B4 OH

(cis); mp.: 215° C. 422 B4 OH

(trans); mp.: 178° C. 423 B3b OCH₃

mp.: 160° C. 424 B3b OCH₃

(cis); mp.: 106° C. 425 B3b OCH₃

(trans); mp.: 120° C. 426 B3b OCH₃

(cis); mp.: 121° C. 427 B3b H

mp.: 156° C. 428 B3b OCH₃

(cis); mp.: 156° C. 429 B3b OCH₃

(trans); mp.: 197° C. 430 B3b CH₃

(B) 431 B3b CH₃

(A)

TABLE 7

Co. Ex. no. no. R¹ L physical data 432 B16

mp.: 128° C. 433 B4

mp.: 175° C. 434 B4

mp.: 170° C. 435 B4

mp.: 103° C. 436 B4

mp.: 151° C. 437 B4

(trans); mp.: 110° C. 438 B4

mp.: 150° C. 439 B4

mp.: 150° C. 440 B4

(cis) 441 B4

mp.: 166° C. 442 B4

mp.: 173° C. 443 B4

mp.: 208° C. 444 B4

mp.: 149° C. 445 B4

mp.: 133° C. 446 B3b

mp.: 150° C. 447 B3b

mp.: 165° C. 448 B3b

mp.: 147° C. 449 B3b

mp.: 154° C. 450 B3b

mp.: 157° C. 451 B4

mp.: 190° C. 452 B4

mp.: 187° C. 453 B3b

mp.: 200° C. 454 B3b

mp.: 160° C. 455 B3b

mp.: 139° C. 456 B3b

(A); mp.: 174° C. 457 B3b

(B); mp.: 160° C. 458 B3b

mp.: 184° C. 459 B4

— 460 B4

mp.: 134° C. 461 B4

(B); mp.: 156° C. 462 B4

mp.: 153° C. 463 B3b

mp.: 161° C. 464 B4

mp.: 135° C. 465 B4

mp.: 131° C. 466 B3b

.HCl(1:1); mp.: 206° C. 467 B3d

mp.: 142° C. 468 B4

.hydrate(1:1); mp.: 104° C. 469 B3b dimethylethyl

mp.: 104° C. 470 B3b

mp.: 161° C. 472 B3b

mp.: 144° C. 473 B4

mp.: 143° C. 474 B4

mp.: 196° C. 475 B4

mp.: 162° C. 476 B4

mp.: 171° C. 477 B4

mp.: 155° C. 478 B2 trimethylmethyl

mp.: 124° C. 479 B4

(A); mp.: 146° C. 480 B4

(B); mp.: 162° C. 481 B4

(A); mp.: 129° C. 482 B4

mp.: 115° C. 483 B2

mp.: 187° C. 484 B2

mp.: 162° C. 485 B4

(A); mp.: 130° C. 486 B4

(A); mp.: 124° C. 487 B4

(B); mp.: 128° C. 488 B4

mp.: 85° C. 489 B2

mp.: 150° C. 490 B4

(A); mp.: 117° C. 491 B2

mp.: 220° C. 492 B4

mp.: 136° C. 493 B2

mp.: 131° C. 494 B4

(A); mp.: 125° C. 495 B4

mp.: 135° C. 496 B4

mp.: 139° C. 497 B4

mp.: 127° C. 498 B16

mp.: 195° C. 499 B2

mp.: 201° C. 500 B3b

mp.: 143° C. 501 B3b

mp.: 137° C. 502 B2

mp.: 210° C. 503 B3d

mp.: 134° C. 504 B2

mp.: 163° C. 505 B4

mp.: 142° C. 506 B2

mp.: 139° C. 507 B4

mp.: 171° C. 512 B3b

— 523 B3b

—

TABLE 8 Co. Ex. no. no. Structure physical data 511 B11

— 514 B12

— 515 B13

— 524 B9a

mp.: 185° C. 471 B15

(E) 526 B14

.HCl(1:1)C. Preparation of Radioactively Labelled CompoundsC.1 [³H]-Labelled Compounds

To a carefully measured amount of palladium on carbon (10%, 0.872 mg)was added a solution of compound 498 (I, 0.919 mg, 2.4 μmol) andtriethylamine (0.92 μl, 6.6 μmol) in sodium-dried tetrahydrofuran (175μl). The reaction flask was connected to a tritiation manifold systemand the reaction mixture was carefully degassed. Tritium gas (19.5 Ci ata pressure of 1017 mbar) was generated from uranium tritide and wasallowed onto the at room temperature stirred reaction mixture. After 30min, the reaction mixture was frozen with liquid nitrogen and the excessof tritium gas was recaptured onto uranium sponge. The solvent waslyophilized from the reaction mixture. Methanol (100 μl) was introducedand lyophilized in order to remove labile tritium. This procedure wasrepeated twice more. The residue was taken up in ethanol, filtered overa GHP Acrodisk 13 mm syringe filter and depleted with ethanol to a totalvolume of 50.0 ml. It contained 71 mCi of radioactivity with[³H]-compound 528 (II) at a 67% radiochemical purity. From this amount,a fraction (5.0 ml) was taken and thoroughly purified in portions viapreparative HPLC (Kromasil KR100-10, column dimensions 4.6 mm ID×300mm). UV detection took place at 265 nm. Elution was performedisocratically with water-methanol-acetonitrile-diisopropylamine(47:26.5:26.5:0.2; v/v/v/v) at a flow rate of 2.0 ml/min. The productcontaining fractions were combined and concentrated under vacuum at 30°C. The residue was dissolved in ethanol (5.0 ml) and concentrated again.This procedure was repeated twice more. The remaining residue wasfinally dissolved in ethanol (20.0 ml) and stored as such. The batchcontained [³H]-compound 528 (II) with a total radioactivity of 3.83 mCiat a purity>98% and at a specific activity of about 25 Ci/mmol.

D. Pharmacological Examples

D1. Signal Transduction at the Cloned Rat mGlu1 Receptor in CHO Cells

CHO cells expressing the mGlu1 receptor were plated in precoated black96-well plates. The next day, the effect of the present compounds onglutamate-activated intracellular Ca²⁺ increase was evaluated in afluorescent based assay. The cells were loaded with Fluo-3 AM, plateswere incubated for 1 hour at room temperature in the dark, cells werewashed and the present compounds were added onto the cells for 20minutes. After this incubation time, the glutamate-induced Ca²⁺ rise wasrecorded for each well in function of time using the Fluorescent ImagePlate Reader (FLIPR, Molecular Devices Inc.). Relative fluorescenceunits were recorded and average data graphs of quadruple wells wereobtained. Concentration-response curves were constructed based on peakfluorescence (maximum signal between 1 and 90 secondes) for eachconcentration of tested compound. pIC₅₀ values are the −log values ofthe concentration of the tested compounds resulting in 50% inhibition ofthe glutamate-induced intracellular Ca²⁺ rise.

The compounds according to the present invention exhibited a pIC₅₀ valueof at least 5.

The compounds that are included in the Tables 1-8 exhibited a pIC₅₀value of at least 6.

A particular group of compounds exhibited a pIC₅₀ value between 7 and 8.It concerns the compounds listed in Table 9. TABLE 9 Com.nr. pIC₅₀ 4637.98 441 7.95 334 7.95 22 7.94 421 7.94 15 7.93 440 7.93 139 7.93 1787.92 338 7.91 87 7.90 462 7.90 394 7.90 423 7.89 21 7.87 220 7.87 4797.86 483 7.86 485 7.84 9 7.84 110 7.84 248 7.84 341 7.83 163 7.81 4337.79 238 7.79 224 7.78 437 7.78 498 7.78 449 7.77 242 7.76 346 7.74 1827.73 486 7.73 447 7.72 7 7.72 175 7.71 475 7.71 480 7.71 213 7.70 2397.70 241 7.67 461 7.65 115 7.64 445 7.63 281 7.63 487 7.63 299 7.63 4317.61 98 7.57 464 7.57 446 7.56 251 7.55 484 7.54 494 7.53 128 7.52 3447.52 161 7.49 298 7.48 454 7.45 456 7.45 277 7.44 91 7.43 356 7.42 2297.41 333 7.41 326 7.41 369 7.40 430 7.39 435 7.38 35 7.36 228 7.36 4297.36 117 7.35 291 7.35 313 7.35 280 7.34 460 7.34 482 7.34 343 7.33 4257.32 473 7.32 287 7.31 448 7.31 243 7.29 323 7.28 159 7.28 289 7.27 1847.26 436 7.26 89 7.25 108 7.25 373 7.25 255 7.23 527 7.23 303 7.22 2967.22 221 7.21 193 7.21 14 7.20 131 7.19 438 7.19 148 7.18 496 7.18 2367.17 332 7.17 481 7.16 191 7.16 457 7.14 20 7.14 145 7.13 268 7.13 5127.13 474 7.13 10 7.11 307 7.11 426 7.11 466 7.10 97 7.08 83 7.08 4347.08 300 7.08 199 7.07 290 7.06 112 7.05 348 7.05 286 7.03 442 7.03 4227.02 283 7.02 318 7.02 36 7.00 396 7.00

A particular group of compounds exhibited a pIC₅₀ value of at least 8.It concern the compounds listed in Table 10. TABLE 10 Comp. nr.Structure pIC50 416

8.587 27

8.527 174

8.49 506

8.48 25

8.45 4

8.4 19

8.38 429

8.38 424

8.355 176

8.33 210

8.315 114

8.28 488

8.27 504

8.27 477

8.25 432

8.237 214

8.233 465

8.145 135

8.14 420

8.135 292

8.13 427

8.115 208

8.095 419

8.065 455

8.055 418

8.045 497

8.025 439

8.023 237

8.01 499

8D2. In Vitro Binding Experiments with a [³H]-Radiolabelled CompoundAccording to the Invention

As [³H]-radiolabelled compounds are used: compound 528, hereafter namedas [³H]Compound A, which is the tritium-radiolabelled equivalent ofcompound 432.

In the next paragraphs, a study will be disclosed illustrating the useof radiolabelled compounds according to the invention.

Materials

All cell culture reagents were obtained from Invitrogen (Carlsbad, USA).Glutamate was obtained from Aldrich Chemical Company (Milwaukee, Wis.);[³H]quisqualate (29 Ci/mmol), [³H]Ro 48-8587 (53 Ci/mmol),myo-[³H]-inositol (22 Ci/mmol) and [³⁵S]GTPγS (1030 Ci/mmol) wereobtained from Amersham (Paisley, UK). [³R]MK-801 (22.5 Ci/mmol) and[³H]CGP39653 (20-50 Ci/mmol) were obtained from NEN (Zaventem, Belgium).GDP was obtained from Boehringer Manheim (Basel, Switzerland) andglycine from BioRad (CA, USA). [³H]L689560 (10-30 Ci/mmol), [³H]LY341495(34.61 Ci/mmol), [³H]MPEP (50.2 Ci/mmol), (S)-4C3HPG, (1S,3R)-ACPD,(S)-3,5-DHPG, (S)-4CPG, AIDA, MCPG, MPEP, CPCCOEt, L-SOP andL-quisqualic acid were purchased from Tocris Cookson (Essex, UK). BAY36-7620, NPS 2390 and phencyclidine were synthesized in-house. Fluo-3-AMand pluronic acid were obtained from Molecular Probes (Leiden, TheNetherlands). Probenecid, strychnine, D-serine and Triton X-100 werepurchased from Sigma-Aldrich (Steinheim, Germany). All other reagentswere from Merck (Darmstadt, Germany).

Cell Transfection and Culture

L929sA cells stably expressing the human mGlu1a receptor were obtainedas described in Lavreysen et al., Mol. Pharmacol. 61:1244-1254, 2002 andwere cultured in Glutamax-I medium supplemented with 10% heatinactivated dialysed foetal calf serum, 0.1 mg/ml streptomycin sulphateand 100 units/ml penicillin. CHO-dhfr⁻ cells stably expressing the ratmGlu1a, -2, -3, -4, -5 and -6 receptor were a kind gift from S.Nakanishi (Tokyo University, Japan) and were grown in DMEM withGlutamax-I with 10% heat inactivated dialysed foetal calf serum, 0.4 mML-prolin, 0.2 mg/ml streptomycin sulphate and 200 units/ml penicillin.Cells were kept in an atmosphere of 37° C. and 5% CO₂.

Intracellular Ca²⁺ Response in Rat and Human mGlu1a Receptor ExpressingCells and in Rat mGlu5 Receptor Expressing Cells

Intracellular calcium ion levels ([Ca²⁺]_(i)) in human mGlu1a receptorexpressing L929sA cells were measured using the Fluorometric ImagingPlate Reader (FLIPR, Molecular Devices, Calif., USA), as described inLavreysen et al., Mol. Pharmacol. 61:1244-1254, 2002. The same procedurewas followed for CHO-dhfr⁻ cells expressing the rat mGlu1a receptor. Forthe rat mGlu5 receptor, cells were seeded at 30.000 cells/well 2 daysbefore the experiment.

IP Response in Rat mGlu1a Receptor Expressing CHO-dhfr⁻Cells

IP accumulation was measured as described in Lavreysen et al., Mol.Pharmacol. 61:1244-1254, 2002. Briefly, cells were seeded at 30,000cells/well in 24-well plates and were labelled with 2.5 μCi/mlmyo-[³H]inositol overnight. On the day of the experiment, cells werewashed and incubated for 10 min with 10 mM LiCl. After 30 min incubationwith increasing concentrations of [³H]Compound A, 1 N HClO₄ was addedand plates were put at 4° C. KOH/phosphate solution and a solutioncontaining 30 mM Na₂B₄O₇.10H₂O and 3 mM EDTA were added prior toapplication to ion exchange chromatography.

Membrane Preparation from CHO-dhfr⁻ Cells Expressing the Rat mGlu1a, -2,-3, -4, -5 and -6 Receptor

Confluent cells were washed in ice-cold phosphate-buffered saline andstored at −20° C. until membrane preparation. After thawing, cells weresuspended in 50 mM Tris-HCl, pH 7.4 and collected through centrifugationfor 10 min at 23,500 g at 4° C. The cells were lysed in 10 mM hypotonicTris-HCl, pH 7.4. After recentrifugation for 20 min at 30,000 g at 4°C., the pellet was homogenized with an Ultra Turrax homogenizer in 50 mMTris-HCl, pH 7.4. Protein concentrations were measured by the Bio-Radprotein assay using bovine serum albumin as standard.

[³⁵S]GTPγS Binding to Membranes from CHO-dhfr⁻ Cells Expressing the RatmGlu2, -3, -4 and -6 Receptor

Membranes were thawed on ice and diluted in 10 mM HEPES acid, 10 mMHEPES salt, pH 7.4, containing 100 mM NaCl, 3 mM MgCl₂, 3 μM GDP and 10μg/ml saponine. Assay mixtures contained 10 μg of membrane protein andwere pre-incubated with compounds or buffer for 5 min at 37° C. Then,glutamate was added and the assay mixtures were further incubated for 30min at 37° C. [³⁵S]GTPγS was added to a final concentration of 0.1 nMfor another 30 min at 37° C. Reactions were terminated by rapidfiltration through Unifilter-96 GF/B filter plates (Packard, Meriden,Conn.) using a 96-well Packard filtermate harvester. Filters were washed2 times with ice-cold 10 mM NaH₂PO₄/10 mM Na₂HPO₄ buffer, pH 7.4.Filter-bound radioactivity was counted in a Microplate Scintillation andLuminesence Counter from Packard.

Radioligand Binding to Rat mGlu1a Receptor CHO-dhfr⁻ Membranes

[³H]Compound A-binding. After thawing, the membranes were homogenizedusing an Ultra Turrax homogenizer and suspended in ice-cold bindingbuffer containing 50 mM Tris-HCl (pH 7.4), 1.2 mM MgCl₂, 2 mM CaCl₂,unless otherwise indicated. Ligand saturation experiments were performedat apparent binding equilibrium (30 min incubation) with 20 μg membraneprotein and 10 concentrations (0.1, 0.2, 0.3, 0.4, 0.5, 1, 2, 2.5, 5 and10 nM) of radioligand. Non-specific binding was estimated in thepresence of 1 μM compound 135. The incubation was stopped by rapidfiltration under suction over GF/C glass-fibre filters using a manual40-well filtration manifold. To measure association kinetics, membraneswere incubated at 4° C., 25° C. or 37° C. in the presence of 2.5 nM[³H]Compound A for 2, 5, 10, 15, 20, 30, 45, 60, 90 or 120 min, thenterminated by rapid filtration using a manual 40-well filtration unit.Dissociation kinetics were measured by adding, at different times beforefiltration 1 μM compound 135 to membranes preincubated for 30 min at 4°C. or 25° C. in the presence of 2.5 nM [³H]Compound A. The filters weretransferred to scintillation vials and, after the addition ofUltima-Gold MV, the radioactivity collected on the filters was countedin a Packard scintillation counter. For inhibition experiments, assaymixtures were incubated for 30 min at 4° C. in a volume of 0.5 mlcontaining 10-20 μg membrane protein, appropriate concentrations of testcompounds and 2.5 nM [³H]Compound A. Non-specific binding was defined asabove. Filtration was performed using Unifilter-96 GF/C filter platesand a 96-well Packard filtermate harvester. After the addition ofmicroscint-O, radioactivity on the filters was counted in a MicroplateScintillation and Luminesence Counter from Packard.

[³H]quisqualate binding. Thawed membranes were homogenized and suspendedin ice-cold binding buffer. For saturation experiments, 30 μg ofmembrane protein was incubated for 1 h at 25° C. with 10 concentrations(1, 2, 5, 10, 20, 40, 60, 90, 120 and 150 nM) of [³H]quisqualate.Non-specific binding was determined in the presence of 1 mM L-glutamate.Bound and free radioligand was separated by rapid filtration over GF/Cglass-fibre filters using a manual 40-well filtration manifold. Forinhibition experiments, 30 μg membrane protein was incubated for 1 h at25° C. in a volume of 0.5 ml containing appropriate concentrations oftest compounds and a final concentration of 10 nM [³H]quisqualate.Filtration was performed using Unifilter-96 GF/C filter plates and aPackard filtermate harvester. Radioactivity trapped on the filters wascounted as above.

Radioligand Binding to Membranes from CHO-dhfr⁻ Cells Expressing the RatmGlu2, -3, -4, -5 and -6 Receptor

After thawing, the membranes were homogenized using an Ultra Turraxhomogenizer and suspended in ice-cold binding buffer containing 50 mMTris-HCl (pH 7.4), 1.2 mM MgCl₂, 2 mM CaCl₂. For [³H]Compound A binding,20 to 160 μg membrane protein and a final concentration of 20 nM[³H]Compound A was used. As indicated in the results section, differentblancs were used to define non-specific binding. Incubation time andtemperature as well as filtration were as described for rat mGlu1areceptor CHO-dhfr⁻ membranes. Expression of rat mGlu2, -3, -5 and mGlu6receptors was confirmed by specific binding of [³H]LY341495 (mGlu2, -3and -6) or [³H]MPEP (mGlu5). For [³H]LY341495 binding, 1 nM (mGlu2 andmGlu3) or 10 nM (mGlu6) [³H]LY341495 was used. Non-specific binding wasdetermined using 1 mM glutamate. Assay mixtures were incubated for 30min (mGlu2 and mGlu3) or 60 min (mGlu6) at 4° C. Incubation was stoppedby filtration over GF/B glass fibre filters (Whatman, England) using amanual 40-well filtration manifold. For rat mGlu5 receptor CHO-dhfr⁻membranes, 10 nM [³H]MPEP and 10 μM MPEP, to reveal non-specificbinding, were used. Incubation was performed at 4° C. for 30 min. Boundand free radioligand were separated over GF/C glass-fibre filters(Whatman, England) using a 40-well filtration unit.

[³H]Compound A Binding to Rat Brain Membranes.

Tissue preparation. Male Wistar rats (˜200 g) were sacrificed bydecapitation. The brains were rapidly removed and cortex, hippocampus,striatum and cerebellum were immediately dissected. The fresh tissue washomogenized with an Ultra Turrax in 20 volumes of 50 mM Tris-HCl, pH 7.4and tissue was centrifuged at 23,500 g for 10 min. After homogenisationusing a DUAL homogeniser, membranes were washed twice by centrifugationat 23,500 g for 10 min. The final pellet was suspended in 10 volumes of50 mM Tris-HCl, pH 7.4 and frozen at −80° C.

In vitro binding assay. After thawing, membranes from rat cortex,cerebellum, striatum and hippocampus were rehomogenized using a DUAL andsuspended in ice-cold binding buffer containing 50 mM Tris-HCl, 1.2 mMMgCl₂, 2 mM CaCl₂, pH 7.4. The binding assay was carried out in a totalvolume of 0.5 ml containing 2.5 nM [³H]Compound A and a membrane aliquotcorresponding to 40 μg for cerebellar membranes, 60 μg for hippocampalmembranes, 80 μg for striatal membranes or 150 μg for corticalmembranes. Specific binding was calculated as the difference between thetotal binding and the binding measured in the presence of 1 μM compound135. After incubation for 30 min at 4° C., the labelled membranes werewashed and harvested by rapid vacuum filtration over Whatman GF/Cglass-fibre filters using a 40-well filtration manifold andradioactivity collected on the filters was counted as above.

[³H]Ro 48-8587, [³H]L689560, [³H]CGP39653 and [³H]MK-801 binding to ratbrain membranes.

Tissue preparation. Male Wistar rats (˜200 g) were sacrificed bydecapitation. The brains were rapidly removed and forebrain wasdissected. The tissue was homogenized with an Ultra Turrax in 20 volumesof ice-cold H₂O and was centrifuged at 48,000 g for 20 min. Afterhomogenisation using a DUAL homogeniser, membranes were washed bycentrifugation at 48,000 g for 10 min. The pellet was then suspended in20 volumes of 50 mM Tris-HCl, pH 7.4 containing 0.04% Triton X-100 andagain centrifuged at 48,000 g for 20 min. The final pellet was frozen at−80° C.

In vitro binding assay. At the day of the experiment, the pellet wasthawed, washed and rehomogenised using a DUAL in ice-cold 50 mMTris-acetate, pH 7.4. Assay conditions for the different radioligandswere as follows. The final concentration of membrane in the assay was 20mg/ml (wet weight) for [³H]Ro 48-8587 and [³H]L689560 and was 10 mg/ml(wet weight) for [³H]CGP39653 and [³H]MK-801. Radioligand concentrationsof 2 nM [³H]Ro 48-8587, 2 nM [³H]L689560, 2 nM [³H]CGP39653 and 3 nM[³H]MK-801 were used. Incubation was performed in the presence of 1 mMKSCN for [³H]Ro 48-8587, 100 μM strychnine for [³H]L689560 and 1 μMglycine+1 μM glutamate for [³H]MK-801 binding. Non-specific binding wasdetermined in the presence of 1 mM glutamate for [³H]Ro 48-8587 and[³H]CGP39653 binding. For [³H]L689560 and [³H]MK-801 binding, 100 μMD-serine or 10 μM phencyclidine were used to define non-specificbinding, respectively. Assays were incubated for 1 h at 37° C., 2 h at4° C., 30 min at 25° C. and 1 h at 4° C. for [³H]Ro 48-8587,[³H]L689560, [3H]CGP39653 and [³H]MK-801 binding, respectively. Afterincubation, bound and free radioligand was separated using a 40-wellfiltration manifold. Radioactivity collected on the filters was countedas above.

[³H]Compound A-Binding and Autoradiography on Rat Brain Sections.

Tissue preparation. Male Wistar rats (200 g) were sacrificed bydecapitation. Brains were immediately removed from the skull and wererapidly frozen in dry-ice-cooled 2-methylbutane (−40° C.). Brains werethen stored at −70° C. until sectioning. Twenty-micrometer-thicksagittal sections were cut using a Leica C3050 cryostat microtome (LeicaMicrosystems, Wetzlar, Germany) and thaw-mounted on SuperFrost Plusmicroscope slides (Menzle-glaser, Germany). The sections were then keptat −70° C. until use.

Receptor autoradiography. Sections were thawed and dried under a streamof cold air, preincubated (3×5 min) in 50 mM Tris-HCl, 1.2 mM MgCl₂, 2mM CaCl₂, 0.1% BSA pH 7.4 at room temperature. Sections were thenincubated for 60 min at room temperature, in buffer containing 50 mMTris-HCl, 1.2 mM MgCl₂, 2 mM CaCl₂, 0.1% BSA (pH 7.4) and 1.5 nM[³H]Compound A. Non-specific binding was determined by addition of 1 μMcompound 135 in the incubation buffer. After the incubation, the excessof radioligand was washed off (3×5 min) in ice-cold buffer containing 50mM Tris-HCl, 1.2 mM MgCl₂ and 2 mM CaCl₂, followed by a rapid dip incold distilled water. The sections were dried under a stream of cold airand then exposed to [³H]Hyperfilm (Amersham, UK) for 6 weeks at roomtemperature. The films were developed manually in Kodak D19 and fixedwith Kodak Readymatic. Some sections were exposed to a Fuji ImagingPlate for 2 days at room temperature and scanned using a Fujix Bass 2000phosphoimager.

Data Analysis and Statistics

Data analysis was performed using the GraphPad Prism program (GraphPadPrism Software, Inc., San Diego, Calif.). Saturation binding experimentswere analysed using a non-linear regression analysis. Inhibition curveswere fitted using non-linear regression analysis fitting the one-sitecompetition equation: Y=Bottom+((Top−Bottom)/1+10^(X-LogIC50)). K_(i)values were calculated using the Cheng-Prusoff equation:K_(i)=IC₅₀/[1+([C]/K_(D))] where C is the concentration of radioligandand K_(D) is the dissociation constant of the radioligand (Cheng andPrusoff, Biochem. Pharmacol. 22, 3099-3108, 1973). The observed on(k_(ob)) and off (k_(off)) rate were calculated fromassociation-dissociation curves using the one-phase-exponentialassociation and decay equations in the Prism program, respectively.k_(on) was calculated by subtracting k_(off) from k_(ob) and dividing bythe radioligand concentration. The two-tailed Student's t-test was usedfor statistical evaluation of the binding data: * p<0.05, ** p<0.01 and*** p<0.001. The Dunnett's t-test following a 2-way analysis of variance(with as factors compound concentration and experiment) were used toanalyse the data from the IP experiments.

Results

Selectivity and mode of antagonism of Compound A for the mGlu1 receptor.In CHO-dhfr⁻ cells expressing the rat mGlu1a receptor, compound Ainhibited the glutamate-induced increase in [Ca²⁺]_(i) with an IC₅₀value of 21.6±5.0 nM (n=4; FIG. 1A) and appeared to be about 8 timesmore potent than the recently described specific mGlu1 receptorantagonist BAY 36-7620 (IC₅₀=161±38 nM, n=3) and 500 times more potentthan CPCCOEt (IC₅₀=10.3±0.8 μM, n=3), tested in the same assay. For thehuman mGlu1a receptor, compound A had an IC₅₀ value of 10.4±4.7 nM(n=3). Compound A did not inhibit glutamate-induced Ca²⁺ signaling ofthe rat mGlu5 receptor expressed in CHO-dhfr⁻ cells, tested up to aconcentration of 10 μM. IC₅₀ values for inhibition of glutamate (30μM)-induced [³⁵S]GTPγS activation were above 30 μM at recombinant ratmGlu2, -3, -4 or -6 receptors. In [³⁵S]GTPγS assays, compound A did notexhibit agonist activity towards any of the mGlu receptors up to aconcentration of 30 μM. In addition, it was investigated whethercompound A could act as a positive allosteric modulator on one of thesemGlu receptor types. For this, we performed glutamateconcentration-response curves by adding glutamate alone or together with10 μM compound A. [³⁵S]GTPγS assays on recombinant rat mGlu2, -3, -4 or-6 receptors showed that the glutamate EC₅₀ was not altered and that theglutamate E_(max) value was not increased upon addition of compound A.The EC₅₀ and E_(max) value of glutamate-induced intracellular Ca²⁺mobilization also did not change in cells expressing the rat mGlu5receptor when compound A was added together with glutamate (data notshown). Together these data exclude agonist, antagonist or positiveallosteric action on mGlu2, -3, -4, -5 and -6 receptors. Radioligandbinding studies on rat forebrain using [³H]Ro-488587, [³H]L689560,[³H]CGP39653 and [³H]MK-801 revealed that compound A did not bind to theAMPA receptor, nor did it bind to the glycine, glutamate or channel poresite of the NMDA receptor (tested up to a concentration of 10 μM),respectively. To analyse how compound A inhibits glutamate activation ofthe mGlu1a receptor, mobilization of Ca²⁺ in response to glutamate wascompared in the absence and presence of compound A (FIG. 1B). Thepresence of compound A not only caused a right-ward shift in theconcentration-response curve of glutamate, but also resulted in adramatic decrease in the maximal response evoked by the agonist,revealing that antagonism by compound A was non-competitive. Completeinhibition of mGlu1a receptor-mediated signalling was observed in thepresence of 100 nM-1 μM compound A. To investigate whether compound Acould act as an inverse agonist, we measured basal IP accumulation inrat mGlu1a receptor containing CHO-dhfr⁻ cells in the presence ofcompound A. FIG. 1C shows that there is a clear reduction in basal IPproduction with increasing concentration of compound A. This reductionwas statistically significant (p<0.05) as off 1 μM compound A, at whichbasal IP accumulation decreased by 24±4%. A maximal decrease of 33±3%was found when using 100 μM compound A. These data indicate thatcompound A can indeed act as an inverse agonist towards the mGlu1areceptor.

Characterization of [³H]Compound A binding to rat mGlu1a receptorCHO-dhfr⁻ membranes. The specific binding of 2.5 nM [³H]Compound A at 4°C. to rat mGlu1a receptor CHO-dhfr⁻ membranes was proportional to theamount of membrane protein and increased linearly between 10 and 50 μgmembrane protein per assay (FIG. 2). Non-specific binding was definedusing 1 μM compound 135 as inhibitor. compound 135 was identified as aspecific mGlu1 receptor antagonist with a potency of 7.2±1.2 nM (n=3)for reversal of glutamate-induced [Ca²⁺]_(i) mobilization. Using 20 μgprotein per assay, specific binding of [³H]Compound A was ˜92% of thetotal binding; in typical assay conditions, total and non-specificbinding were in the range of 3,800 and 300 DPM, respectively.

Addition of 1.2 mM MgCl₂ and 2 mM CaCl₂ caused a slight increase inspecific binding (data not shown). Further addition of NaCl (10-100-300mM) had no effect. While specific binding decreased by 22% at pH 6,increasing the pH up to 10 had no effect (data not shown).

Association kinetics were measured as described in Materials andMethods.

Decreasing the incubation temperature to 4° C., dramatically enhancedspecific binding, whereas a low binding was found at 37° C. (FIG. 3).The association of [³H]Compound A to membranes was extremely fast. At 4°C., 2 min incubation resulted already in a specific bindingcorresponding to about 70% of the quantity bound at equilibrium. Maximalbinding was reached within 5 min incubation for each incubationtemperature. Analysis of the association curves resulted in observedassociation rate constants (k_(ob)) of 0.6285, 2.571 and 1.523 min⁻¹ at4° C., 25° C. and 37° C., respectively. The kinetics of dissociationwere also rapid (FIG. 4). At 25° C., the radioligand dissociated withinas little as 2 min after 1 μM compound 135 was added to the reactiontubes. The rapid dissociation kinetics at 25° C. did not allow us tocalculate an accurate dissociation rate constant (k_(off)). Dissociationoccurred more gradual when incubated at 4° C. [³H]Compound A wasdisplaced completely within approximately 45 min after the addition ofan excess compound 135. Analysis of the dissociation curve at 4° C.resulted in an k_(off) of 0.1249 min⁻¹ k_(on)(k_(ob)−k_(off)/radioligand concentration) at 4° C. was 0.1007 nM⁻¹min⁻¹.

Ligand saturation experiments were performed at apparent bindingequilibrium (30 min incubation) and with 10 concentrations ofradioligand. FIG. 5 shows the saturation curve and Scatchard Plot of[³H]Compound A binding to rat mGlu1a receptor CHO-dhfr⁻ membranes.Scatchard Plots were linear, indicating the presence of a single,saturable, high affinity binding site. Non-linear regression analysis ofthe rectangular hyperbola revealed a B_(max) of 6512±1501 fmoles/mg ofprotein and a K_(D) of 0.90±0.14 nM (n=3).

A series of mGlu1 receptor agonists and antagonists was tested forinhibition of [³H]Compound A-binding to rat mGlu1a receptor CHO-dhfr⁻membranes. Inhibition curves for some antagonists are shown in FIG. 6,and the K_(i) values of all compounds tested are listed in Table 11.TABLE 11 Potencies of various mGlu1 receptor agonists and antagonists ininhibition of [³H]Compound A-binding to rat mGlu1a receptor CHO-dhfr⁻membranes. K_(i) values and Hill coefficients are mean ± SD of 3-4independent experiments. compound K_(i) (nM) Hill coefficient Compound A1.35 ± 0.99 0.94 ± 0.04 NPS 2390 1.36 ± 0.50 0.97 ± 0.02 BAY 36-762011.2 ± 0.93 0.95 ± 0.02 CPCCOEt 4,900 ± 170   0.93 ± 0.03glutamate >1,000,000 quisqualate >1,000,000 1S,3R-ACPD >1,000,000(S)-3,5-DHPG >1,000,000 LY367385 >1,000,000 (S)-4C3HPG >1,000,000AIDA >1,000,000 (S)-4CPG >1,000,000 MCPG >1,000,000

Remarkably, all ligands that bind to the glutamate binding site, i.e.glutamate, quisqualate, 1S,3R-ACPD, (S)-3,5-DHPG, LY-367385,(S)-4C-3HPG, (S)-4CPG, MCPG and AIDA did not inhibit [³H]Compound Abinding. In contrast, the non-competitive mGlu1 receptor antagonistsCPCCOEt, BAY 36-7620, NPS 2390 and compound A inhibited [³H]Compound Abinding to rat mGlu1a receptor CHO-dhfr⁻ membranes with potencies,generally consistent with their potencies to inhibit mGlu1a receptorfunction. compound A and NPS 2390 showed the highest affinity, with aK_(i) of 1.35±0.99 and 1.36±0.50 nM, respectively. BAY 36-7620 inhibitedthe binding also at nanomolar concentrations, whereas CPCCOEt displacedat micromolar concentrations.

We also investigated the specificity of [³H]Compound A binding towardsthe mGlu1 versus mGlu2, -3, -4, -5, and -6 receptors. Using[³H]LY341495, 95, 98 and 40% specific binding was found when usingmembranes prepared from CHO-dhfr⁻ cells expressing the mGlu2, mGlu3, ormGlu6 receptor, respectively. [³H]MPEP was used as a positive controlfor the mGlu5 receptor and produced 95% specific binding to rat mGlu5receptor containing membranes. Total binding of 20 nM [³H]Compound A tomembranes prepared from CHO-dhfr⁻ cells expressing the rat mGlu2, -3,-4, -5, or -6 receptor was not higher than the binding to membranes fromwild-type CHO-dhfr⁻ cells, nor was it higher than the non-specificbinding to rat mGlu1a receptor CHO-dhfr⁻ membranes. Furthermore,specific binding of [³H]Compound A to these membranes was investigatedusing various blancs: 1 μM compound 135, which is expected to bind tothe same site as compound A, glutamate and L-SOP, which bind to theglutamate binding pocket and MPEP that binds to an allosteric site onthe mGlu5 receptor (see Table 12). None of these blancs displaced[³H]Compound A. Together, these data demonstrate the specificity of [³H]Compound A for the mGlu1 receptor relative to mGlu2, -3, -4, -5 and -6receptor subtypes. TABLE 12 [³H]Compound A is specific for the mGlu1receptor relative to the mGlu2, −3, −4, −5 or −6 receptor. The specificbinding of 20 nM [³H]Compound A to 40 μg membranes from wild-type(CHO-dhfr⁻) cells or from CHO-dhfr⁻ cells expressing rat mGlu2, −3, −4,−5, or −6 receptors is compared to binding of 10 nM [³H]Compound A to 20μg rat mGlu1a receptor CHO-dhfr⁻ membranes. Various compounds were usedto define non-specific binding. Specific binding (SB) data from ratmGlu1a receptor CHO-dhfr⁻ membranes are the mean ± SD of 3 experimentsperformed in duplicate. Other SB data are the mean of duplicatedeterminations from one experiment (ND = not determined). SB (fmoles/mgwild- protein) mGlu1 type mGlu2 mGlu3 mGlu4 mGlu5 mGlu6 Compound 135 as6057 ± 65  0 0 82 38 0 blanc 1456 ND  34^(a)  0^(a)  57^(b)   0^(c) 0^(a) various blancs ND^(a)1 mM glutamate was used to determine non-specific binding^(b)0.1 mM L-SOP was used to determine non-specific binding^(c)10 μM MPEP was used to determine non-specific bindingComparison with [³H]quisqualate binding. Saturation binding experimentswere performed using 30 μg protein per incubate and 10 concentrations(1, 2, 5, 10, 20, 40, 60, 90, 120 and 150 nM) of the mGlu1 receptoragonist [³H]quisqualate (FIG. 7). Fitting of the curves revealed asingle binding site with K_(D) and B_(max) values of 22.0±10 nM and3912±436 fmoles/mg protein, respectively (n=3). Clearly, [³H]Compound Abound to mGlu1a with a much higher affinity than [³H]quisqualate does.The number of binding sites labelled with [³H]quisqualate was −60% ofthe number of binding sites labelled by [³H]Compound A.

The same compounds were evaluated for their inhibitory action on[³H]quisqualate binding to rat mGlu1a receptor CHO-dhfr⁻ membranes.Inhibitory potencies of the tested agonists and antagonists as well asHill coefficients are summarized in Table 13. In this case, thecompounds known to exert a competitive interaction with glutamate,inhibited [³H]quisqualate binding, whereas CPCCOEt, BAY 36-7620 and NPS2390 did not affect [³H]quisqualate binding. Also compound A did notdisplace binding of [³H]quisqualate to the rat mGlu1a receptor. Thecompetitive mGlu1 receptor ligands displaced [³H]quisqualate bindingwith the following rank order of potency:quisqualate>glutamate>LY367385>(S)-3,5-DHPG>(S)-4C-3HPG>1S,3R-ACPD>(S)-4CPG>AIDA>MCPG.TABLE 13 Potencies of various mGlu1 receptor agonists and antagonists ininhibition of [³H]quisqualate binding to rat mGlu1a receptor CHO-dhfr⁻membranes. K_(i) values and Hill coefficients are mean ± SD of 2independent experiments. compound K_(i) (μM) Hill coefficientquisqualate 0.030 ± 0.00  0.98 ± 0.01 glutamate 0.40 ± 0.07 0.99 ± 0.01LY367385 1.18 ± 0.47 0.99 ± 0.01 (S)-3,5-DHPG 1.42 ± 0.00 0.97 ± 0.00(S)-4C3HPG 1.65 ± 0.06 0.98 ± 0.02 1S,3R-ACPD 1.92 ± 0.20 0.97 ± 0.01(S)-4CPG 4.51 ± 0.78 0.98 ± 0.00 AIDA 98.3 ± 15.4 0.98 ± 0.03 MCPG  165± 0.47 0.96 ± 0.02 Compound A >1,000 NPS 2390 >1,000 BAY 36-7620 >1,000CPCCOEt >1,000

Nature of competition between CPCCOEt, BAY 36-7620, NPS 2390 and[³H]Compound A binding. The fact that the non-competitive compounds alldisplaced [³H]Compound A binding without affecting the binding of[³H]quisqualate suggested that these antagonists bound another site thanthe glutamate binding site. In order to assess whether the referencecompounds CPCCOEt, BAY 36-7620, NPS 2390 and the newly identified mGlu1receptor antagonist compound A compete for the same site or mutuallyexclusive sites, saturation experiments with [³H]Compound Aconcentrations from 0.2 to 20 nM in the absence and the presence ofCPCCOEt (30 μM), BAY 36-7620 (100 nM) and NPS 2390 (10 nM) wereperformed. The presence of these competitors did not affect the B_(max)values, but caused a significant increase in the K_(D) value of[³H]Compound A (Table 14). This is visualized in FIG. 8, where the dataare plotted using linear regression. In Scatchard plots, the obtainedlinear lines indeed merge to the same intercept on the X-axis (i.e. theB_(max) value). TABLE 14 K_(D) and B_(max) values obtained from analysesof [³H]Compound A saturation binding curves obtained in the absence andpresence of the mGlu1 receptor antagonists CPCCOEt (30 μM), BAY 36-7620(100 nM) and NPS 2390 (10 nM). Values are mean ± SD from 3 individualexperiments. Statistical analysis was performed using the Student'st-test (two-tailed): **p < 0.01 and ***p < 0.001. control CPCCOEt BAY36-7620 NPS 2390 K_(D) (nM) 0.73 ± 0.09  3.17 ± 0.90**  5.21 ± 1.2**  2.90 ± 0.20*** B_(max) (fmoles/mg 7284 ± 970  7009 ± 1231 5872 ± 10186887 ± 2804 protein)[³H]Compound A binding in rat brain membranes and sections. We used thespecific mGlu1 receptor radioligand [³H]Compound A to examine receptorbinding in different regions of the rat brain. Membranes from ratcortex, striatum, cerebellum and hippocampus were prepared and[³H]Compound A binding was measured. Non-specific binding compared tototal binding was 10% in cerebellum, 30% in hippocampus and 25% incortex and striatum. K_(D) and B_(max) values were determined for eachbrain region (Table 15). K_(D) values were about 1 nM for allstructures. The B_(max) values were significantly different among thevarious areas. [³H]Compound A labelled a remarkably high number of mGlu1receptors in the cerebellum. In the striatum and hippocampus about 16%of the number of sites found in the cerebellum were labelled. Only 11%of the number of binding sites in the cerebellum was bound in the ratcortex. Importantly, also incubation with 10 μM of the structurallyunrelated compound BAY 36-7620 maximally inhibited [³H]Compound Abinding (FIG. 9). The mGlu5 receptor selective compound MPEP (tested upto 30 μM) did not affect [³H]Compound A binding to rat cerebellarmembranes, again showing the mGlu1 receptor selectivity of compound A.

Using radioligand autoradiography, we examined [³H]Compound A bindingdistributions in rat brain sections in further detail (FIG. 10).[³H]Compound A autoradiography was investigated in sagittal rat brainsections; non-specific binding was determined using compound 135 (FIG.10, panel A). Very high specific binding was observed in the molecularlayer of the cerebellum. A moderate signal was observed in the CA3 fieldand dentate gyrus of the hippocampal formation, thalamus, olfactorytubercle, amygdala and substantia nigra reticulata. The cerebral cortex,caudate putamen, ventral pallidum, nucleus accumbens showed lowerlabelling. Also incubation with BAY 36-7620 completely inhibited[³H]Compound A binding to rat brain sections (FIG. 10, panel C). TABLE15 Equilibrium binding constants of [³H]Compound A-binding to membranesfrom rat cortex, hippocampus, striatum and cerebellum. K_(D) and B_(max)values are mean ± SD derived from 3 independent experiments. cortexhippocampus striatum cerebellum K_(D) (nM) 1.04 ± 0.40 0.72 ± 0.22 0.84± 0.23 0.99 ± 0.36 B_(max)(fmoles/ 471 ± 68  688 ± 125 741 ± 48  4302 ±2042 mg protein)Discussion

Up to now, only a few mGlu1 receptor subtype selective antagonists havebeen found. The mGlu1 receptor has been shown to be selectively blockedby CPCCOEt (Litschig et al., Mol. Pharmacol. 55:453-461, 1999) and BAY36-7620 with potencies that vary from micromolar for CPCCOEt (6.6 μM) tohigh nanomolar concentrations for BAY 36-7620 (160 nM). In the presentstudy, compound A is identified as a novel mGlu1 receptor antagonistwith low nanomolar functional antagonistic potency on the rat mGlu1areceptor (21.6 nM) and the human mGlu1a receptor (10.4 nM). Theantagonist action of compound A was found to be non-competitive, sincethe maximal glutamate-induced mGlu1 receptor activation was decreased inthe presence of compound A. The observed increase in glutamate EC₅₀ inthe presence of compound A can be explained by the presence of sparereceptors. In the presence of low concentrations of a non-competitiveantagonist, the concentrations-response curve will be shifted to theright since more agonist is needed to compensate for the ‘nonspare’receptors that are blocked by the antagonist. These antagonistconcentrations will not yet affect the maximal agonist response, whilehigher antagonist concentrations will eventually suppress the maximumresponse (Zhu et al., J. Pharm. Tox. Meth. 29:85-91, 1993). Thisphenomenon has also been reported for BAY 36-7620 (Carroll et al., Mol.Pharmacol. 59:965-973, 2001) and CPCCOEt (Hermans et al.,Neuropharmacology 37:1645-1647, 1998). Our data further show thatcompound A may act as an inverse agonist towards the mGlu1a receptor andthat compound A acts selectively on the mGlu1 receptor with regard toother mGlu receptor subtypes and ionotropic glutamate receptors. Signaltransduction data showed that compound A does not display agonist,antagonist or positive allosteric action on the mGlu2, -3, -4, -5 and -6receptor and radioligand binding studies revealed that [³H]Compound Adoes not bind to the mGlu2, -3, -4, -5 and -6 receptor, furthermoreexcluding the possibility that compound A acts as a neutral ligand atany of these receptor types. The lack of selective mGlu1 receptorradioligands together with the interesting pharmacological properties ofcompound A were compelling reasons to label compound A for theinvestigation of mGlu1 receptors in binding studies.

[³H]Compound A binding met all the requirements for a ligand very wellsuited to study binding properties, pharmacology and distribution ofmGlu1 receptors. First, [³H]Compound A binding studies were performed inrat mGlu1a receptor CHO-dhfr⁻ membranes. Specific binding was very highand increased linearly with protein concentration (FIG. 2). Specificbinding showed a modest increase in the presence of MgCl₂ and CaCl₂,whereas binding decreased by 22% at pH 6 and was unaffected by anincrease in pH. In regard to the effects of pH on binding, it is worthnoting the calculated physicochemical properties of compound A:calculated pK_(a) and clogP are 6.2 and 4.5, respectively. At pH 7.4,the degree of ionisation of compound A is thus very low (only 5.9%). Thepercentage of ionisation decreases further at higher pH (1.6% at pH 8,0.2% at pH 9 and no protonation at pH 10). The clogD value remains 4.5from pH 7.4 to pH 10. At pH 6, however, 61.3% of compound A is in theprotonated form. Accordingly, the clogD decreases to 4.1. The lowerbinding of the ligand in ionised form suggests that the non-ionisedligand has the highest binding affinity. This is remarkable, and is incontrast with findings for ligands for mono-amine G protein-coupledreceptors (e.g. the dopamine receptor), which are often strong bases andbind in a cationic form. For such compounds, the driving force for thebinding to the receptor is electrostatic in nature (Van de Waterbeemd etal., J. Med. Chem. 29:600-606, 1986). Our data may indicate that ionicinteractions are not a driving force in the binding to the receptor, andthat there is neither a contribution of ionic surface effects.Additionally, although compound A is a strong lipophilic compound, thevery low non-specific [³H]Compound A binding might be due to the factthat no electrostatic interaction can take place between the non-ionisedform of compound A and the negatively charged cell membrane. Binding wastemperature dependent, and increased substantially at 4° C. (FIG. 3). Byvirtue of its fast association and dissociation kinetics, bindingequilibrium was rapidly reached. [³H]Compound A labelled apparently asingle population of sites with a very high affinity (K_(D)=0.90±0.14nM). In contrast, [³H]quisqualate, the mGlu1 receptor radioligand ofchoice up to now, exhibited a much higher K_(D) value of 22.0±10 nM,which correlated well with the value of 37 nM obtained by Mutel et al.,J. Neurochem, 75:2590-2601, 2000. Besides the considerable higheraffinity, [³H]Compound A labelled significantly more (˜40%) bindingsites than [³H]quisqualate. B_(max) values of 6512±1501 fmoles/mg ofprotein and 3912±436 fmoles/mg protein were found for [³H]Compound A and[³H]quisqualate, respectively. This discrepancy can be explained on thebasis of the G protein coupling of the receptor. Agonists facilitate thecoupling of the receptor to the G protein, which results in a receptorconformation with high affinity for agonists. According to this theory,a full agonist such as quisqualate would predominantly label the highaffinity or G protein-coupled receptor state. An antagonist would haveequal affinity for coupled and uncoupled receptors, and thus for boththe high and low affinity states of the receptor. Our finding that theB_(max) for [³H]Compound A is considerably higher than for[³H]quisqualate is in line with this theory.

A striking finding in this study was that the natural agonist glutamateand also quisqualate were unable to inhibit [³H]Compound A binding torat mGlu1a receptor CHO-dhfr⁻ membranes, whereas CPCCOEt, BAY 36-7620,NPS 2390 and compound A, known as non-competitive antagonists, allinhibited [³H]Compound A binding to the same maximal level (FIG. 6).Inhibition of [³H]Compound A binding by the latter compounds followedsigmoidal curves with Hill coefficients of about 1.0 (Table 11), whichgave no indication for binding to multiple sites. It is important tomention that although a structurally related analogue was used to definenon-specific binding, a similar low non-specific binding was obtainedwith structurally unrelated compounds such as BAY 36-7620 when used at 1μM or more in rat mGlu1a receptor CHO-dhfr⁻ membranes (FIG. 6). For[³H]quisqualate, all the amino acid-like structures, known ascompetitive ligands, could displace [³H]quisqualate from its bindingsite. Inhibitory potencies of quisqualate, glutamate, LY367385,(S)-3,5-DHPG, (S)-4C-3HPG, 1S, 3R-ACPD, (S)-4CPG, AIDA and MCPG (Table13) were in good agreement with the values reported by Mutel et al. J.Neurochem. 75:2590-2601, 2000. In contrast, the above non-competitivecompounds did not affect its binding. For CPCCOEt, it has been reportedthat it does not affect [³H]glutamate binding to membranes prepared fromrat mGlu1a receptor-expressing cells (Litschig et al., Mol. Pharmacol.55:453-461, 1999). Furthermore, it has been suggested that CPCCOEt doesnot bind to the glutamate binding site, but interacts with Thr815 andAla818 in transmembrane domain VII. CPCCOEt is proposed to interferewith receptor signalling by disrupting an intramolecular interactionbetween the glutamate-bound extracellular domain and the transmembranedomain VII. Caroll et al. in Mol. Pharmacol. 59:965-973, 2001demonstrated that BAY 36-7620 did not displace [³H]quisqualate from theglutamate binding pocket. Transmembrane helices 4 to 7 were shown toplay a crucial role for binding of BAY 36-7620. Our inhibitionexperiments performed with [³H]Compound A and [³H]quisqualate suggestthat CPCCOEt, BAY 36-7620, NPS 2390 bind to the same site as compound A.Saturation experiments using [³H]Compound A in the absence and thepresence of 30 μM CPCCOEt, 100 nM BAY 36-7620 and 10 nM NPS 2390 furthersupport that these compounds bind to the same or mutually exclusivesites. K_(D) values significantly increased, whereas the B_(max) valuewas unaltered (Table 14). These results indicate that although theaffinity of [³H]Compound A decreases, high concentrations of[³H]Compound A are still able to displace binding of the anothercompound from its binding site, which is a typical property of acompetitive interaction. In conclusion, our data support the notion thatCPCCOEt, BAY 36-7620, NPS 2390 and compound A act on a site differentfrom the glutamate binding pocket, presumably they compete for the sametransmembrane segment VII.

Previous group I mGlu receptor binding studies in brain were performedusing [³H]glutamate or [³H]quisqualate (Schoepp and True, Neurosci. Lett145:100-104, 1992; Wright et al., J. Neurochem. 63:938-945, 1994; Mutelet al., J. Neurochem. 75:2590-2601, 2000). These radioligands have thedisadvantage of labelling more than one type of glutamate receptor.Therefore, selective inhibitors had to be added to the incubation bufferto prevent labelling to other metabotropic or ionotropic glutamatereceptor subtypes. To date, there is no radioligand available tospecifically study the binding and distribution of the mGlu1 receptor.The specific mGlu1 receptor labelling of [³H]Compound A makes itparticularly useful for the investigation of native mGlu1 receptors inrat or human brain. Experiments using rat cortex, hippocampus, striatumand cerebellum membranes revealed that [³H]Compound A specific binding,defined in the presence of 1 μM compound 135, was high, especially inthe cerebellum (only 10% non-specific binding). Saturation experimentsshowed that [³H]Compound A again labelled apparently a single bindingsite with very high affinity. K_(D) values of about 1 nM were found forall the different brain areas (Table 15). A striking difference inB_(max) values was found: a large population of binding sites waslabelled in the cerebellum, whereas in hippocampus, striatum and cortexmoderate to low levels of receptor expression were detected.

Because of its specificity, [³H]Compound A proved to be particularlysuitable for investigation of mGlu1 receptor distribution in brainsections using radioligand autoradiography. mGlu1 receptorautoradiography revealed that the highest level of mGlu1 specificbinding was present in the molecular layer of the cerebellum. Thegranule cell layer was very weakly labelled. These results were alsofound by Mutel et al. in J. Neurochem. 75:2590-2601, 2000 whoinvestigated group I mGlu receptor distribution using [³H]quisqualate.In the hippocampal formation, the CA3 dendritic field together with themolecular layer of the dentate gyrus showed abundant labelling. The CA1area showed very weak [³H]Compound A binding, corresponding well withimmunohistochemistry data from Lujan et al., Eur. J. Neurosci.8:1488-1500, 1996 and Shigemoto et al., J. Neurosci. 17:7503-7522, 1997,who showed that in CA1 dendritic fields, an antibody specific for themGlu5 receptor but not a specific mGlu1 receptor antibody yieldedintense immunolabelling. Autoradiography experiments using[³H]quisqualate indeed revealed staining in both the CA1 and CA3 regionof the hippocampus, indicating binding to both the mGlu1 and mGlu5receptor, respectively (Mutel et al., J. Neurochem. 75:2590-2601, 2000).[³H]Compound A binding was also quite high in the thalamus, olfactorytubercle, amygdala and substantia nigra reticulata and was somewhatlower in the cerebral cortex, caudate putamen, nucleus accumbens andventral pallidum. The same structures were labelled using[³H]quisqualate (Mutel et al., J. Neurochem. 75:2590-2601, 2000). Alsoimmunocytochemical findings on the cellular localization of the mGlu1areceptor, using an antibody selective for the mGlu1a receptor, weregenerally consistent with our data (Martin et al., Neuron. 9:259-270,1992). Since [³H]Compound A is expected to label all mGlu1 receptorsplice variants known to date, the distribution of 1 splice variant mayhowever differ from that of our radiolabel. For example, in the CA3region and the caudate putamen, which are labelled by the radioligand,mGlu1b receptor but no or little mGlu1a receptor immunoreactivity wasfound (Martin et al., Neuron. 9:259-270, 1992; Shigemoto et al., J.Neurosci. 17:7503-7522, 1997; Ferraguti et al., J. Comp. Neur.400:391-407, 1998). An important point in the demonstration of theidentity of the [³H]Compound A-labeled sites was the finding that thestructurally different compound BAY 36-7620 also fully displaced[³H]Compound A binding to rat brain membranes (FIG. 9) as well as tobrain sections (FIG. 10), providing a good guarantee that the inhibitedbinding is purely receptor specific and not related to a structuralmoiety of the radioligand.

In this application, we have shown that [³H]Compound A is an excellentradioligand to study mGlu1 receptors in an heterologous expressionsystem, rat brain homogenates and brain sections. We can conclude thatbecause of its minimal non-specific binding, its high binding affinityand marked selectivity, [³H]Compound A is the ligand of choice forfurther exploration of the mGlu1 receptor. [³H]Compound A opensperspectives for a detailed investigation of subcellular and cellularlocalization of the mGlu1 receptor and for the study of the functionalrole and regulation of the receptor in various areas.

LIST OF FIGURES

FIG. 1: Antagonist profile of Compound A. Inhibition of glutamate (30μM)-induced Ca²⁺ mobilization in CHO-dhfr⁻ cells expressing the ratmGlu1a receptor is shown in FIG. 1A. Data are expressed as percentage ofthe signal obtained using 30 μM glutamate, which was set at 100% and aremean±SD of 3 experiments. FIG. 1B shows a concentration-response curveof glutamate alone or together with 20 nM, 30 nM, 60 nM, 100 nM and 1 μMCompound A. Values are mean±SD of triplicate determinations within 1experiment. An additional experiment showed the same results. FIG. 1Cshows basal IP accumulation in the presence of increasing concentrationsof Compound A. Values are expressed as percentage of basal IP productionin the presence of solvent, which was set as 100% and are mean±SD of 3experiments performed in quadruplicate.

FIG. 2: Specific [³H]Compound A binding is linear with amount ofmembrane protein. 10 to 50 μg rat mGlu1a receptor CHO-dhfr⁻ membraneswas incubated for 30 min on ice with 2.5 nM [³H]Compound A. Data areexpressed as mean±SD of triplicate determinations and are from arepresentative experiment.

FIG. 3: Association time-course curve for [³H]Compound A binding to ratmGlu1a receptor CHO-dhfr⁻ membranes. Association kinetics were measuredby adding 2.5 nM [³H]Compound A at different times before filtration andwas determined at 3 different temperatures. Data are mean±SD of 3independent experiments performed in duplicate.

FIG. 4: Time-course for dissociation of [³H]Compound A to rat mGlu1areceptor CHO-dhfr⁻ membranes at 4° C. and 25° C. Samples were incubatedfor 30 min at 4° C. or 25° C., then an excess of compound 135 was added,followed by rapid filtration at the time indicated for each data point.Values are mean±SD of 2 independent experiments performed in duplicate.

FIG. 5: Representative saturation binding curve and Scatchard Plot of[³H]Compound A binding to rat mGlu1a receptor CHO-dhfr⁻ membranes.Specific binding (SB) was obtained by calculating the difference betweentotal binding (TB) and non-specific binding (BL), measured in thepresence of 1 μM compound 135. For each experiment, data points weredetermined in duplicate. The experiment was repeated 3 times.

FIG. 6: Inhibition of 2.5 nM [³H]Compound A binding to rat mGlu1areceptor CHO-dhfr⁻ membranes by various mGlu receptor antagonists. Datapoints represent % of total binding and are mean±SD of 3-4 individualexperiments.

FIG. 7: Representative saturation binding curve and Scatchard Plot of[³H]quisqualate binding to rat mGlu1a receptor CHO-dhfr⁻ membranes. Foreach experiment, data points were determined in duplicate. Theexperiment was repeated 2 times.

FIG. 8: Saturation binding curves and Scatchard plots of [³H]Compound Abinding to rat mGlu1a receptor CHO-dhfr⁻ membranes in the absence andpresence of CPCCOEt (30 μM), BAY 36-7620 (100 nM) and NPS 2390 (10 nM).The graph shown is a representative of 3 independent experiments. Dataare expressed in nM specifically bound. For each experiment, data pointswere determined in duplicate.

FIG. 9: Inhibition of 2.5 nM [³H]Compound A binding to rat cerebellarmembranes by BAY 36-7620. Data points represent % of total binding andare mean±SD of 2 individual experiments.

FIG. 10: [³H]Compound A binding to sagittal rat brain sections usingautoradiography. Panel A is a representative section showing totalbinding with 1.5 nM [³H]Compound A. Panel B is a representative andadjacent section showing non-specific binding with 1.5 nM [³H]Compound Ain the presence of 1 μM compound 135. Panel C is a representativesection showing non-specific binding with 1.5 nM [³H]Compound A in thepresence of 10 μM BAY 36-7620. Sections from panel A and B were exposedto [³H]Hyperfilm, while the section from panel C was exposed to a FujiImaging plate. Th, thalamus; SNr, substantia nigra reticulata; CA3, CA3region of the hippocampus; Dg; dentate gyrus of the hippocampus; Cer;cerebellum; Cp, caudate putamen; Cx, cerebral cortex, Ot, olfactorytubercle, Am, amygdala, Vp, ventral pallidum, Na, nucleus accumbens.

1. A radiolabelled compound according to Formula (I-A)* or (I-B)*

an N-oxide form, a pharmaceutically acceptable addition salt, aquaternary amine and a stereochemically isomeric form thereof, wherein Xrepresents O; C(R⁶)₂ with R⁶ being hydrogen, aryl or C₁₋₆alkyloptionally substituted with amino or mono- or di(C₁₋₆alkyl)amino; S orN—R⁷ with R⁷ being amino or hydroxy; R¹ represents C₁₋₆alkyl; aryl;thienyl; quinolinyl; cycloC₃₋₁₂alkyl or (cycloC₃₋₁₂alkyl)C₁₋₆alkyl,wherein the cycloC₃₋₁₂alkyl moiety optionally may contain a double bondand wherein one carbon atom in the cycloC₃₋₁₂alkyl moiety may bereplaced by an oxygen atom or an NR⁸-moiety with R⁸ being hydrogen,benzyl or C₁₋₆alkyloxycarbonyl; wherein one or more hydrogen atoms in aC₁₋₆alkyl-moiety or in a cycloC₃₋₁₂alkyl-moiety optionally may bereplaced by C₁₋₆alkyl, hydroxyC₁₋₆alkyl, haloC₁₋₆alkyl, aminoC₁₋₆alkyl,hydroxy, C₁₋₆alkyloxy, arylC₁₋₆alkyloxy, halo, C₁₋₆alkyloxycarbonyl,aryl, amino, mono- or di(C₁₋₆alkyl)amino, C₁₋₆alkyloxycarbonylamino,halo, piperazinyl, pyridinyl, morpholinyl, thienyl or a bivalent radicalof formula —O—, —O—CH₂—O or —O—CH₂—CH₂—O—; or a radical of formula (a-1)

wherein Z₁ is a single covalent bond, O, NH or CH₂; Z₂ is a singlecovalent bond, O, NH or CH₂; n is an integer of 0, 1, 2 or 3; andwherein each hydrogen atom in the phenyl ring independently mayoptionally be replaced by halo, hydroxy, C₁₋₆alkyl, C₁₋₆alkyloxy orhydroxyC₁₋₆alkyl; or X and R¹ may be taken together with the carbon atomto which X and R¹ are attached to form a radical of formula (b-1), (b-2)or (b-3);

R² represents hydrogen; halo; cyano; C₁₋₆alkyl; C₁₋₆alkyloxy;C₁₋₆alkylthio; C₁₋₆alkylcarbonyl; C₁₋₆alkyloxycarbonyl;C₁₋₆alkylcarbonyloxyC₁₋₆alkyl; C₂₋₆alkenyl; hydroxyC₂₋₆alkenyl;C₂₋₆alkynyl; hydroxyC₂₋₆alkynyl; tri(C₁₋₆alkyl)silaneC₂₋₆alkynyl; amino;mono- or di(C₁₋₆alkyl)amino; mono- or di(C₁₋₆alkyloxyC₁₋₆alkyl)amino;mono- or di(C₁₋₆alkylthioC₁₋₆alkyl)amino; aryl; arylC₁₋₆alkyl;arylC₂₋₆alkynyl; C₁₋₆alkyloxyC₁₋₆alkylaminoC₁₋₆alkyl; aminocarbonyloptionally substituted with C₁₋₆alkyl, C₁₋₆alkyloxyC₁₋₆alkyl,C₁₋₆alkyloxycarbonylC₁₋₆alkyl or pyridinylC₁₋₆alkyl; a heterocycleselected from thienyl, furanyl, pyrrolyl, thiazolyl, oxazolyl,imidazolyl, isothiazolyl, isoxazolyl, pyrazolyl, pyridyl, pyrazinyl,pyridazinyl, pyrimidinyl, piperidinyl and piperazinyl, optionallyN-substituted with C₁₋₆alkyloxyC₁₋₆alkyl, morpholinyl, thiomorpholinyl,dioxanyl or dithianyl; a radical —NH—C(═O)R⁹ wherein R⁹ representsC₁₋₆alkyl optionally substituted with cycloC₃₋₁₂alkyl, C₁₋₆alkyloxy,C₁₋₆alkyloxycarbonyl, aryl, aryloxy, thienyl, pyridinyl, mono- ordi(C₁₋₆alkyl)amino, C₁₋₆alkylthio, benzylthio, pyridinylthio orpyrimidinylthio; cycloC₃₋₁₂alkyl; cyclohexenyl; amino;arylcycloC₃₋₁₂alkylamino; mono- or -di(C₁₋₆alkyl)amino; mono- ordi(C₁₋₆alkyloxycarbonylC₁₋₆alkyl)amino; mono- ordi(C₁₋₆alkyloxycarbonyl)amino; mono- or di(C₂₋₆alkenyl)amino; mono- ordi(arylC₁₋₆alkyl)amino; mono- or diarylamino; arylC₂₋₆alkenyl;furanylC₂₋₆alkenyl; piperididinyl; piperazinyl; indolyl; furyl;benzofuryl; tetrahydrofuryl; indenyl; adamantyl; pyridinyl; pyrazinyl;aryl; arylC₁₋₆alkylthio or a radical of formula (a-1); a sulfonamid—NH—SO₂—R¹⁰ wherein R¹⁰ represents C₁₋₆alkyl, mono- or polyhaloC₁₋₆alkyl, arylC₁₋₆alkyl, arylC₂₋₆alkenyl, aryl, quinolinyl,isoxazolyl or di(C₁₋₆alkyl)amino; R³ and R⁴ each independently representhydrogen; halo; hydroxy; cyano; C₁₋₆alkyl; C₁₋₆alkyloxy;C₁₋₆alkyloxyC₁₋₆alkyl; C₁₋₆alkylcarbonyl; C₁₋₆alkyloxycarbonyl;C₂₋₆alkenyl; hydroxyC₂₋₆alkenyl; C₂₋₆alkynyl; hydroxyC₂₋₆alkynyl;tri(C₁₋₆alkyl)silaneC₂₋₆alkynyl; amino; mono- or di(C₁₋₆alkyl)amino;mono- or di(C₁₋₆alkyloxyC₁₋₆alkyl)amino; mono- ordi(C₁₋₆alkylthioC₁₋₆alkyl)amino; aryl; morpholinylC₁₋₆alkyl orpiperidinylC₁₋₆alkyl; or R² and R³ may be taken together to form—R²—R³—, which represents a bivalent radical of formula —(CH₂)₃—,—(CH₂)₄—, —(CH₂)₅—, —(CH₂)₆—, —CH═CH—CH═CH—, -Z₄-CH═CH—, —CH═CH-Z₄-,-Z₄-CH₂—CH₂—CH₂—, —CH₂-Z₄-CH₂—CH₂—, —CH₂—CH₂-Z₄-CH₂—, —CH₂—CH₂—CH₂-Z₄-,-Z₄-CH₂—CH₂—, —CH₂-Z₄-CH₂— or —CH₂—CH₂-Z₄-, with Z₄ being O, S, SO₂ orNR¹¹ wherein R¹¹ is hydrogen, C₁₋₆alkyl, benzyl or C₁₋₆alkyloxycarbonyl;and wherein each bivalent radical is optionally substituted withC₁₋₆alkyl. or R³ and R⁴ may be taken together to form a bivalent radicalof formula —CH═CH—CH═CH— or —CH₂—CH₂—CH₂—CH₂—; R⁵ represents hydrogen;cycloC₃₋₁₂alkyl; piperidinyl; oxo-thienyl; tetrahydrothienyl,arylC₁₋₆alkyl; C₁₋₆alkyloxyC₁₋₆alkyl; C₁₋₆alkyloxycarbonylC₁₋₆alkyl orC₁₋₆alkyl optionally substituted with a radical C(═O)NR_(x)R_(y), inwhich R_(x) and R_(y), each independently are hydrogen, cycloC₃₋₁₂alkyl,C₂₋₆alkynyl or C₁₋₆alkyl optionally substituted with cyano,C₁₋₆alkyloxy, C₁₋₆alkyloxycarbonyl, furanyl, pyrrolidinyl, benzylthio,pyridinyl, pyrrolyl or thienyl; Y represents O or S; or Y and R⁵ may betaken together to form ═Y—R⁵— which represents a radical of formula—CH═N—N═  (c-1);—N═N—N═  (c-2); or—N—CH═CH—  (c-3); aryl represents phenyl or naphthyl optionallysubstituted with one or more substituents selected from halo, hydroxy,C₁₋₆alkyl, C₁₋₆alkyloxy, phenyloxy, nitro, amino, thio, C₁₋₆alkylthio,haloC₁₋₆alkyl, polyhaloC₁₋₆alkyl, polyhaloC₁₋₆alkyloxy,hydroxyC₁₋₆alkyl, C₁₋₆alkyloxyC₁₋₆alkyl, aminoC₁₋₆alkyl, mono- ordi(C₁₋₆alkyl)amino; mono- or di(C₁₋₆alkyl)aminoC₁₋₆alkyl, cyano,—CO—R¹², —CO—OR¹³, —NR¹³SO₂R¹², —SO₂—NR¹³R¹⁴, —NR¹³C(O)R¹²,—C(O)NR¹³R¹⁴, —SOR¹², —SO₂R¹²; wherein each R¹², R¹³ and R¹⁴independently represent C₁₋₆alkyl; cycloC₃₋₆alkyl; phenyl; phenylsubstituted with halo, hydroxy, C₁₋₆alkyl, C₁₋₆alkyloxy, haloC₁₋₆alkyl,polyhaloC₁₋₆alkyl, furanyl, thienyl, pyrrolyl, imidazolyl, thiazolyl oroxazolyl; and when the R¹—C(═X) moiety is linked to another positionthan the 7 or 8 position, then said 7 and 8 position may be substitutedwith R¹⁵ and R¹⁶ wherein either one or both of R¹⁵ and R¹⁶ representsC₁₋₆alkyl, C₁₋₆alkyloxy or R¹⁵ and R¹⁶ taken together may form abivalent radical of formula —CH═CH—CH═CH—.
 2. A radiolabelled compoundaccording to claim 1, wherein X represents O; C(R⁶)₂ with R⁶ beinghydrogen or aryl; or N—R⁷ with R⁷ being amino or hydroxy; R¹ representsC₁₋₆alkyl, aryl; thienyl; quinolinyl; cycloC₃₋₁₂alkyl or(cycloC₃₋₁₂alkyl)C₁₋₆alkyl, wherein the cycloC₃₋₁₂alkyl moietyoptionally may contain a double bond and wherein one carbon atom in thecycloC₃₋₁₂alkyl moiety may be replaced by an oxygen atom or anNR⁸-moiety with R⁸ being benzyl or C₁₋₆alkyloxycarbonyl; wherein one ormore hydrogen atoms in a C₁₋₆alkyl-moiety or in a cycloC₃₋₁₂alkyl-moietyoptionally may be replaced by C₁₋₆alkyl, haloC₁₋₆alkyl, hydroxy,C₁₋₆alkyloxy, arylC₁₋₆alkyloxy, halo, aryl, mono- or di(C₁₋₆alkyl)amino,C₁₋₆alkyloxycarbonylamino, halo, piperazinyl, pyridinyl, morpholinyl,thienyl or a bivalent radical of formula —O— or —O—CH₂—CH₂—O—; or aradical of formula (a-1)

wherein Z₁ is a single covalent bond, O or CH₂; Z₂ is a single covalentbond, O or CH₂; n is an integer of 0, 1, or 2; and wherein each hydrogenatom in the phenyl ring independently may optionally be replaced by haloor hydroxy; or X and R¹ may be taken together with the carbon atom towhich X and R¹ are attached to form a radical of formula (b-1), (b-2) or(b-3);

R² represents hydrogen; halo; cyano; C₁₋₆alkyl; C₁₋₆alkyloxy;C₁₋₆alkylthio; C₁₋₆alkylcarbonyl; C₁₋₆alkyloxycarbonyl; C₂₋₆alkenyl;hydroxyC₂₋₆alkenyl; C₂₋₆alkynyl; hydroxyC₂₋₆alkynyl;tri(C₁₋₆alkyl)silaneC₂₋₆alkynyl; amino; mono- or di(C₁₋₆alkyl)amino;mono- or di(C₁₋₆alkyloxyC₁₋₆alkyl)amino; mono- ordi(C₁₋₆alkylthioC₁₋₆alkyl)amino; aryl; arylC₁₋₆alkyl; arylC₂₋₆alkynyl;C₁₋₆alkyloxyC₁₋₆alkylaminoC₁₋₆alkyl; aminocarbonyl optionallysubstituted with C₁₋₆alkyloxycarbonylC₁₋₆alkyl; a heterocycle selectedfrom thienyl, furanyl, thiazolyl and piperidinyl, optionallyN-substituted with morpholinyl or thiomorpholinyl; a radical —NH—C(═O)R⁹wherein R⁹ represents C₁₋₆alkyl optionally substituted withcycloC₃₋₁₂alkyl, C₁₋₆alkyloxy, C₁₋₆alkyloxycarbonyl, aryl, aryloxy,thienyl, pyridinyl, mono- or di(C₁₋₆alkyl)amino, C₁₋₆alkylthio,benzylthio, pyridinylthio or pyrimidinylthio; cycloC₃₋₁₂alkyl;cyclohexenyl; amino; arylcycloC₃₋₁₂alkylamino; mono- or-di(C₁₋₆alkyl)amino; mono- or di(C₁₋₆alkyloxycarbonylC₁₋₆alkyl)amino;mono- or di(C₁₋₆alkyloxycarbonyl)amino; mono- or di(C₂₋₆alkenyl)amino;mono- or di(arylC₁₋₆alkyl)amino; mono- or diarylamino; arylC₂₋₆alkenyl;furanylC₂₋₆alkenyl; piperididinyl; piperazinyl; indolyl; furyl;benzofuryl; tetrahydrofuryl; indenyl; adamantyl; pyridinyl; pyrazinyl;aryl or a radical of formula (a-1); a sulfonamid —NH—SO₂—R¹⁰ wherein R¹⁰represents C₁₋₆alkyl, mono- or poly haloC₁₋₆alkyl, arylC₁₋₆alkyl oraryl; R³ and R⁴ each independently represent hydrogen; C₁₋₆alkyl;C₁₋₆alkyloxyC₁₋₆alkyl; C₁₋₆alkyloxycarbonyl; or R² and R³ may be takentogether to form —R²—R³—, which represents a bivalent radical of formula—(CH₂)₄—, —(CH₂)₅—, -Z₄-CH═CH—, -Z₄-CH₂—CH₂—CH₂— or -Z₄-CH₂—CH₂—, withZ₄ being O, S, SO₂ or NR¹¹ wherein R¹¹ is hydrogen, C₁₋₆alkyl, benzyl orC₁₋₆alkyloxycarbonyl; and wherein each bivalent radical is optionallysubstituted with C₁₋₆alkyl; or R³ and R⁴ may be taken together to form abivalent radical of formula —CH═CH—CH═CH— or —CH₂—CH₂—CH₂—CH₂—; R⁵represents hydrogen; piperidinyl; oxo-thienyl; tetrahydrothienyl,arylC₁₋₆alkyl; C₁₋₆alkyloxycarbonylC₁₋₆alkyl or C₁₋₆alkyl optionallysubstituted with a radical C(═O)NR_(x)R_(y), in which R_(x) and R_(y),each independently are hydrogen, cycloC₃₋₁₂alkyl, C₂₋₆alkynyl orC₁₋₆alkyl optionally substituted with cyano, C₁₋₆alkyloxy orC₁₋₆alkyloxycarbonyl; Y represents O or S; or Y and R⁵ may be takentogether to form ═Y—R⁵— which represents a radical of formula—CH═N—N═  (c-1); or—N═N—N═  (c-2); aryl represents phenyl or naphthyl optionallysubstituted with one or more substituents selected from halo,C₁₋₆alkyloxy, phenyloxy, mono- or di(C₁₋₆alkyl)amino and cyano; and whenthe R¹—C(═X) moiety is linked to another position than the 7 or 8position, then said 7 and 8 position may be substituted with R¹⁵ and R¹⁶wherein either one or both of R¹⁵ and R¹⁶ represents C₁₋₆alkyl or R¹⁵and R¹⁶ taken together may form a bivalent radical of formula—CH═CH—CH═CH—.
 3. A radiolabelled compound according to claim 1,wherein, X represents O; R¹ represents C₁₋₆alkyl; cycloC₃₋₁₂alkyl or(cycloC₃₋₁₂alkyl)C₁₋₆alkyl, wherein one or more hydrogen atoms in aC₁₋₆alkyl-moiety or in a cycloC₃₋₁₂alkyl-moiety optionally may bereplaced by C₁₋₆alkyloxy, aryl, halo or thienyl; R² represents hydrogen;halo; C₁₋₆alkyl or amino; R³ and R⁴ each independently representhydrogen or C₁₋₆alkyl; or R² and R³ may be taken together to form—R²—R³—, which represents a bivalent radical of formula -Z₄-CH₂—CH₂—CH₂—or -Z₄-CH₂—CH₂— with Z₄ being O or NR¹¹ wherein R¹¹ is C₁₋₆alkyl; andwherein each bivalent radical is optionally substituted with C₁₋₆alkyl;or R³ and R⁴ may be taken together to form a bivalent radical of formula—CH₂—CH₂—CH₂—CH₂—; R⁵ represents hydrogen; Y represents O; and arylrepresents phenyl optionally substituted with halo.
 4. A radiolabelledcompound according to claim 1, wherein, the R¹—C(═X) moiety is linked tothe quinoline or quinolinone moiety in position
 6. 5. A radiolabelledcompound according to claim 1, wherein the compound contains at leastone radioactive atom.
 6. A radiolabelled compound according to claim 5,wherein the radioactive isotope is selected from the group of ³H, ¹¹Cand ¹⁸F.
 7. A radiolabelled compound according to claim 6, wherein thecompound is any one of compounds (a), (b), (c), (d) and (e):


8. A radiolabelled compound according to claim 6, wherein, the compoundis compound (a).
 9. Radioactive composition for the administration tomamals comprising a therapeutically effective amount of a radiolabelledcompound according to claim 1 and a pharmaceutically acceptable carrieror diluent.
 10. A radiolabelled compound according to claim 1 for use ina diagnostic method.
 11. A radiolabelled compound according to claim 1wherein the diagnostic method consists of marking or identifying a mGlu1receptor in biological material.
 12. A radiolabelled compound accordingto claim 1, wherein the marking consists of administering theradiolabelled compound to biological material and the identifyingconsists of detecting the emissions from the radiolabelled compound. 13.A radiolabelled compound according to claim 1, wherein the diagnosticmethod consists of screening whether a test compound has the ability tooccupy or bind to a mGlu1 receptor in biological material.
 14. Aradiolabelled compound or composition according to claim 1 wherein thebiological material is selected from the group of tissue samples, plasmafluids, body fluids, body parts and organs originating from warm-bloodedanimals and warm-blooded animals per se, in particular humans.
 15. Aradiolabelled compound according to claim 1 for the manufacture of adiagnostic tool for marking or identifying an mGlu1 receptor inbiological material.
 16. Use of a radiolabelled compound or compositionaccording to claim 15, wherein the marking consists of administering theradiolabelled compound to biological material and the identifyingconsists of detecting the emissions from the radiolabelled compound. 17.A radiolabelled compound according to claim 1 for the manufacture of adiagnostic tool for screening whether a test compound has the ability tooccupy or bind to a mGlu1 receptor in biological material
 18. (canceled)19. (canceled)
 20. (canceled)